Compositions and methods for liver growth and liver protection

ABSTRACT

The present invention provides pharmaceutical compositions and methods for liver proliferation and protection. Specifically useful are VEGFR modulating agents capable of promoting liver growth. Disclosed compositions and methods may be useful for promoting proliferation or treating pathological conditions in other organs of significant biological functions.

[0001] This is a non-provisional application claiming priority toprovisional application No. 60/386,637, filed Jun. 5, 2002, the entiredisclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the diagnostic and therapeuticuses of VEGFR modulating agents, including methods of utilizing VEGFRagonists for promoting liver growth, treating liver pathologicalconditions, and protecting liver from damage.

BACKGROUND OF THE INVENTION

[0003] Liver

[0004] The liver is the major metabolic control organ of the human bodythat comprises thousands of minute lobules (lobuli hepatis), thefunctional units of the organ. Liver tissue contains two majordifferentiated cell types: parenchymal cells (i.e., hepatocytes) andnon-parenchymal cells. The complex functions of liver are exerted to alarge extent by hepatocytes, whereas non-parenchymal cells such asKupffer cells, Ito cells and liver sinusoidal endothelial cells (LSEC)play important roles in supporting and providing supplies tohepatocytes. Mochida et al. (1996) Biochem. Biophy. Res. Comm.226:176-179.

[0005] The liver acts as a guardian interposed between the digestivetract and the rest of the body. A major hepatic function involveseffective uptake, storage, metabolism and distribution to blood and bilelarge amounts of substances such as carbohydrates, lipids, amino acids,vitamins and trace elements. Another function of the liver is thedetoxification of xenobiotic pollutants, drugs and endogenousmetabolites, through both phase I (oxidation/reduction) and phase II(conjugation) mechanisms.

[0006] Because of its essential role to life, liver dysfunction anddiseases are often debilitating and life threatening. A number of acuteor chronic pathological conditions are associated with structural and/orfunctional abnormalities of the liver. These include, but are notlimited to, liver failure, hepatitis (acute, chronic or alcohol), livercirrhosis, toxic liver damage, medicamentary liver damage, hepaticencephalopathy, hepatic coma or hepatic necrosis.

[0007] Many chemical and biological agents, either therapeutic or purelyharmful, can induce liver damages and thus are hepatotoxic. Thesusceptibility of the liver to damage by hepatotoxic agents may berelated to its primary role in metabolism or is a consequence ofhypersensitivity reactions. Up to 25% of cases of fulminant hepaticfailure may be the result of adverse reactions to medical agents.Hepatotoxic compounds are also an important cause of chronic liverdisease including fatty liver, hepatitis, cirrhosis and vascular andneoplastic lesions of the liver. (Sinclair et al., Textbook of InternalMedicine, 569-575 (1992) (editor, Kelley; Publisher, J. B. LippincottCo.).

[0008] Hepatotoxic agents may induce liver damage by cytotoxicity to theliver directly or through the production of toxic metabolites (thiscategory includes the hypersensitivity reaction which mimics a drugallergy); cholestasis, an arrest in the flow of bile due to obstructionof the bile ducts; and vascular lesions, such as in veno occlusivedisease (VOD), where injury to the vascular endothelium results inhepatic vein thrombosis. Individual susceptibility to liver damageinduced by hepatotoxic agents is influenced by genetic factors, age,sex, nutritional status, exposure to other drugs, and systemic diseases(Sinclair et al., Textbook of Internal Medicine, Supra).

[0009] In addition to normal growth during early development, livertissue has a unique ability to regenerate at adult stage. Liverregeneration after the loss of hepatic tissue is a fundamental componentof the recovery process in response to various forms of liver injurysuch as hepatotoxicity, viral infection, vascular injury and partialhepatectomy. Following partial hepatectomy, for example, the liver sizeis usually restored to its original mass within about six days. Livergrowth and regeneration involves proliferation of both hepatocytes andnon-parenchymal cells such as sinusoidal endothelial cells. Typically,hepatocytes are the first to proliferate, and other cells of the liverenter into DNA synthesis about 24 hours after the hepatocytes.Michalopoulos and DeFrances (1997) Science 276:60-65.

[0010] Factors for liver proliferation

[0011] Several growth factors and cytokines have been implicated asbeing able to induce liver regeneration, most notably hepatocyte growthfactor (HGF), epidermal growth factor (EGF), transforming growthfactor-α (TGF-α), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α),basic and acidic fibroblast growth factors, CTGF, HB-EGF, andnorepinephrine. Fujiwara et al. (1993) Hepatol. 18:1443-9; Baruch et al.(1995) J. Hepatol. 23:328-32; Ito et al. (1994) Biochem. Biophys. Res.Commun. 198:25-31; Suzuma et al. (2000) J. Biol. Chem. 275:40725-31;Michalopoulos and DeFrances (1997) supra. As one of the most potentliver mitogens, HGF was first identified as a factor capable ofstimulating DNA synthesis in cultured hepatocytes but is now known tohave multiple distinct functions on a variety of epithelial cells.Nakamura et al. (1984) Biochem. Biophys. Res. Comm. 122:1450; Russell etal. (1984) J. Cell. Physiol. 119:183-192. Scatter factor (SF), whichenhances motility and invasiveness of certain cell types, was found tohave identical amino acid sequence as HGF, leading to the designationHGF/SF. Stoker and Perryman (1985) J. Cell Sci. 77:209-223; Gherardi andStoker (1990) Nature 346:228. HGF/SF is synthesized as an inactive,single-chain zymogen that is subsequently cleaved to produce an active,dimeric glycoprotein composed of a 69-kDa α-subunit and a 34-kDaβ-subunit held together by a single disulfide bond. Nakamura et al.(1989) Nature 342:440-443; Roos et al. (1995) Am. J. Physiol.268:G380-6.

[0012] All known biological effects of HGF are transduced via a singletyrosine kinase receptor, Met, the product of the Met protooncogene.HGF/SF acts predominantly on Met-expressing epithelial cells in anendocrine and/or paracrine fashion, to mediate such diverse biologicalactivities as proliferation, branching, cell migration, morphogenesisand lumen formation. van der Voort et al. Adv. Cancer Res. 79:39-90(2000). In the liver, HGF is expressed in non-hepatocyte cells such asIto cells and LSECs, whereas met transcripts are strongly expressed inhepatocytes. Hu et al. Am. J. Pathol. 142:1823-1830 (1993). Afterchemical or mechanical liver injury, HGF levels sharply increase,leading to a strong hepatocyte proliferation. Horimoto et al. J.Hepatol. 23:174-183 (1995). Livers from transgenic mice withliver-specific overexpression of HGF are twice the size of livers ofcontrol animals and they regenerate much faster after partialhepatectomy. Sakata et al. (1996) Cell Growth Differ. 7:1513-1523;Shiota et al. (1994) Hepatol. 19:962-972. Furthermore, HGF null mutantmouse embryos fail to develop a fully functional liver, demonstratingthe essential role of HGF during liver development. Schmidt et al.(1995) Nature 373:699-702. The continuous infusion of large doses (5mg/kg/day) of HGF directly into the portal vein has been shown to resultin a significant increase of relative liver mass in mice. Patijn et al.(1998) Hepatol. 28:707-16. While HGF was found to be a potent inducer ofhepatocyte mitosis, however, it failed to induce proliferation ofnonparenchymal cells including sinusoidal endothelial cells. Patijn etal., supra. In other biological contexts, conversely, HGF has been shownas a potent endothelial cell mitogen. Rosen and Goldberg (1997) In:Regulation of Angiogenesis. Rosen, E, Goldberg, ID, Eds. SpringerVerlag. pp 193-208.

[0013] It has been suggested that substantially high HGF plasmaconcentrations may be required in order to promote liver growth in vivo(Roos et al. (1995) Am. J. Physiol. 268:G380-6). HGF, by virtue of itsstrong heparin-binding properties, is largely sequestered inextrahepatic tissues following intravenous administration (Zioncheck etal. (1994) Endocrinology 134:1879-87) and the co-administration ofdextran sulfate is required for an effective liver-promoting action(Roos et al., 1995).

[0014] Angiogenesis and Liver

[0015] Angiogenesis is an important cellular event in which vascularendothelial cells proliferate, prune and reorganize to form new vesselsfrom preexisting vascular network. There are compelling evidences thatthe development of a vascular supply is essential for normal andpathological proliferative processes (Folkman and Klagsbrun (1987)Science 235:442-447). Delivery of oxygen and nutrients, as well as theremoval of catabolic products, represent rate-limiting steps in themajority of growth processes occurring in multicellular organisms. Thus,it has been generally assumed that the vascular compartment isnecessary, albeit but not sufficient, not only for organ development anddifferentiation during embryogenesis, but also for wound healing andreproductive functions in the adult. However, recent evidence suggeststhat, at least in the mouse embryo, the vascular endothelium may have aninductive effect on liver (Matsumoto et al. (2001) Science 294:559-563)and pancreas organogenesis (Lammert et al. (2001) Science 294:564-567),even prior to the establishment of a blood flow. The mechanism of suchinduction is unknown.

[0016] Angiogenesis is also implicated in the pathogenesis of a varietyof disorders, including but not limited to, proliferative retinopathies,age-related macular degeneration, tumors, rheumatoid arthritis (RA), andpsoriasis. Folkman (1995) Nat Med 1:27-31. Regenerating liver, inanalogy to rapidly growing tumors, must synthesize new stroma and bloodvessels. Not surprisingly, therefore, many studies have focused onangiogenesis in liver development and regeneration, as well as the rolesof many known angiogenic factors therein. Michalopoulos and DeFrances(1997) supra; Mochida et al. (1996).

[0017] Vascular endothelial cell growth factor (VEGF), a potent mitogenfor vascular endothelial cells, has been reported as a key regulator ofangiogenesis and vasculogenesis. Ferrara and Davis-Smyth (1997)EndocrineRev. 18:4-25; Ferrara (1999) J. Mol. Med. 77:527-543. Compared to othergrowth factors that contribute to the processes of vascular formation,VEGF is unique in its high specificity for endothelial cells within thevascular system. Recent evidence indicates that VEGF is essential forembryonic vasculogenesis and angiogenesis. Carmeliet et al. (1996)Nature 380:435-439; Ferrara et al. (1996) Nature 380:439-442.Furthermore, VEGF is required for the cyclical blood vesselproliferation in the female reproductive tract and for bone growth andcartilage formation. Ferrara et al. (1998) Nature Med. 4:336-340; Gerberet al. (1999) Nature Med. 5:623-628.

[0018] In addition to being an angiogenic factor in angiogenesis andvasculogenesis, VEGF, as a pleiotropic growth factor, exhibits multiplebiological effects in other physiological processes, such as endothelialcell survival, vessel permeability and vasodilation, monocyte chemotaxisand calcium influx. Ferrara and Davis-Smyth (1997), supra. Moreover,recent studies have reported mitogenic effects of VEGF on a fewnon-endothelial cell types, such as retinal pigment epithelial cells,pancreatic duct cells and Schwann cells. Guerrin et al. (1995) J. CellPhysiol. 164:385-394; Oberg-Welsh et al. (1997) Mol. Cell. Endocrinol.126:125-132; Sondell et al. (1999) J. Neurosci. 19:5731-5740.

[0019] Substantial evidence also implicates VEGF's critical role in thedevelopment of conditions or diseases that involve pathologicalangiogenesis. The VEGF mRNA is overexpressed by the majority of humantumors examined (Berkman et al. J Clin Invest 91:153-159 (1993); Brownet al. Human Pathol. 26:86-91 (1995); Brown et al. Cancer Res.53:4727-4735 (1993); Mattern et al. Brit. J. Cancer. 73:931-934 (1996);and Dvorak et al. Am J. Pathol. 146:1029-1039 (1995)). Also, theconcentration of VEGF in eye fluids are highly correlated to thepresence of active proliferation of blood vessels in patients withdiabetic and other ischemia-related retinopathies (Aiello et al. N.Engl. J. Med. 331:1480-1487 (1994)). Furthermore, recent studies havedemonstrated the localization of VEGF in choroidal neovascular membranesin patients affected by AMD (Lopez et al. Invest. Ophtalmo. Vis. Sci.37:855-868 (1996)). Anti-VEGF neutralizing antibodies suppress thegrowth of a variety of human tumor cell lines in nude mice (Kim et al.Nature 362:841-844 (1993); Warren et al. J. Clin. Invest. 95:1789-1797(1995); Borgström et al. Cancer Res. 56:4032-4039 (1996); and Melnyk etal. Cancer Res. 56:921-924 (1996)) and also inhibit intraocularangiogenesis in models of ischemic retinal disorders (Adamis et al.Arch. Ophthalmol. 114:66-71 (1996)). Therefore, anti-VEGF monoclonalantibodies or other inhibitors of VEGF action are promising candidatesfor the treatment of solid tumors and various intraocular neovasculardisorders.

[0020] Human VEGF was obtained by first screening a cDNA libraryprepared from human cells, using bovine VEGF cDNA as a hybridizationprobe. Leung et al. (1989) Science, 246:1306. One cDNA identifiedthereby encodes a 165-amino acid protein having greater than 95%homology to bovine VEGF; this 165-amino acid protein is typicallyreferred to as human VEGF (hVEGF) or VEGF₁₆₅. The mitogenic activity ofhuman VEGF was confirmed by expressing the human VEGF cDNA in mammalianhost cells. Media conditioned by cells transfected with the human VEGFcDNA promoted the proliferation of capillary endothelial cells, whereascontrol cells did not. Leung et al. (1989) Science, supra.

[0021] Although a vascular endothelial cell growth factor could beisolated and purified from natural sources for subsequent therapeuticuse, the relatively low concentrations of the protein in follicularcells and the high cost, both in terms of effort and expense, ofrecovering VEGF proved commercially unavailing. Accordingly, furtherefforts were undertaken to clone and express VEGF via recombinant DNAtechniques. (See, e.g., Ferrara (1995) Laboratory Investigation72:615-618 (1995), and the references cited therein).

[0022] VEGF is expressed in a variety of tissues as multiple homodimericforms (121, 145, 165, 189, and 206 amino acids per monomer) resultingfrom alternative RNA splicing. VEGF₁₂₁ is a soluble mitogen that doesnot bind heparin; the longer forms of VEGF bind heparin withprogressively higher affinity. The heparin-binding forms of VEGF can becleaved in the carboxy terminus by plasmin to release a diffusibleform(s) of VEGF. Amino acid sequencing of the carboxy terminal peptideidentified after plasmin cleavage is Arg₁₁₀-Ala₁₁₁. Amino terminal“core” protein, VEGF (1-110) isolated as a homodimer, binds neutralizingmonoclonal antibodies (such as the antibodies referred to as 4.6.1 and3.2E3.1.1) and soluble forms of VEGF receptors with similar affinitycompared to the intact VEGF₁₆₅ homodimer.

[0023] Several molecules structurally related to VEGF have also beenidentified recently, including placenta growth factor (PIGF), VEGF-B,VEGF-C, VEGF-D and VEGF-E. Ferrara and Davis-Smyth (1987) Endocr. Rev.,supra; Ogawa et al. (1998) J. Biological Chem. 273:31273-31281; Meyer etal. (1999) EMBO J., 18:363-374. A receptor tyrosine kinase, Flt-4(VEGFR-3), has been identified as the receptor for VEGF-C and VEGF-D.Joukov et al. (1996) EMBO. J. 15:1751; Lee et al. (1996) Proc. Natl.Acad. Sci. USA 93:1988-1992; Achen et al. (1998) Proc. Natl. Acad. Sci.USA 95:548-553. VEGF-C has recently been shown to be involved in theregulation of lymphatic angiogenesis. Jeltsch et al. (1997) Science276:1423-1425.

[0024] Two VEGF receptors have been identified, Flt-1 (also calledVEGFR-1) and KDR (also called VEGFR-2). Shibuya et al. (1990) Oncogene8:519-527; de Vries et al. (1992) Science 255:989-991; Terman et al.(1992) Biochem. Biophys. Res. Commun. 187:1579-1586. Neuropilin-1 hasbeen shown to be a selective VEGF receptor, able to bind theheparin-binding VEGF isoforms (Soker et al. (1998) Cell 92:735-45). BothFlt-1 and KDR belong to the family of receptor tyrosine kinases (RTKs).The RTKs comprise a large family of transmembrane receptors with diversebiological activities. At present, at least nineteen (19) distinct RTKsubfamilies have been identified. The receptor tyrosine kinase (RTK)family includes receptors that are crucial for the growth anddifferentiation of a variety of cell types (Yarden and Ullrich, Ann.Rev. Biochem. 57:433-478, 1988; Ullrich and Schlessinger, Cell61:243-254, 1990). The intrinsic function of RTKs is activated uponligand binding, which results in phosphorylation of the receptor andmultiple cellular substrates, and subsequently in a variety of cellularresponses (Ullrich & Schlessinger, 1990, Cell 61:203-212). Thus,receptor tyrosine kinase mediated signal transduction is initiated byextracellular interaction with a specific growth factor (ligand),typically followed by receptor dimerization, stimulation of theintrinsic protein tyrosine kinase activity and receptortrans-phosphorylation. Binding sites are thereby created forintracellular signal transduction molecules and lead to the formation ofcomplexes with a spectrum of cytoplasmic signaling molecules thatfacilitate the appropriate cellular response. (e.g., cell division,differentiation, metabolic effects, changes in the extracellularmicroenvironment) see, Schlessinger and Ullrich, 1992, Neuron 9:1-20.Structurally, both Flt-1 and KDR have seven immunoglobulin-like domainsin the extracellular domain, a single transmembrane region, and aconsensus tyrosine kinase sequence which is interrupted by akinase-insert domain. Matthews et al. (1991) Proc. Natl. Acad. Sci. USA88:9026-9030; Terman et al. (1991) Oncogene 6:1677-1683.

[0025] There are compelling evidences suggesting that Flt-1 and KDR havedifferent signal transduction properties and possibly mediate differentfunctions. Moreover, the signals mediated through Flt-1 and KDR appearto be cell type specific. Recent studies have provided considerableexperimental data indicating that KDR is the major mediator of themitogenic, angiogenic and permeability-enhancing effects of VEGF(Ferrara (1999) Kidney Int. 56:794-814). VEGF stimulation leads to arobust auto-phosphorylation of KDR and activation of the MAPK cascade,which may directly contribute to endothelial cell proliferation (Krolland Waltenberger (1997) J. Biol. Chem. 272:32521-7). In contrast, thefunction of VEGFR-1 has been less clear, and many apparently conflictingreports on its function exist in the literature. This molecule displaysa very weak or undetectable tyrosine autophosphorylation in endothelialcells in response to VEGF (Gille et al. (2000) EMBO J. 19:4064-4073).Flt-1 has been shown to have inhibitory effects on endothelialmitogenesis in several biological contexts, including early embryonicdevelopment, either by acting as a “decoy” receptor that prevents VEGFbinding to VEGFR-2 or by directly inhibiting VEGFR-2 activities. Park etal. (1994) J. Biol. Chem. 269:25646-54; U.S. Pat. No. 6,107,046 (Alitaloet al.); Fong et al. (1999) Development 126:3015-25; Zeng et al. (2001).J. Biol. Chem. 276:26969-79). Other studies suggest that VEGFR-1 maymediate recruitment of monocytes and endothelial cell progenitors to thetumor vasculature (Barleon et al. (1996) Blood 87:3336-43) (Lyden et al.(2001) Nat. Med. 7:1194-201). Thus, the importance of VEGFR-1 signalingin the vascular endothelium is largely unclear.

[0026] Recent studies have attempted to elucidate the molecularmechanisms of various physiological and pathological processes in theliver, particularly liver regeneration. There have been proposed tworeciprocal paracrine communication systems existing in hepatic tissuesbetween hepatocytes and non-parenchymal cells such as sinusoidalendothelial cells. In one direction, growth factors such as HGF/SF arereleased from non-parenchymal cells such as sinusoidal endothelial cellsand Kupfer cells, bind to their receptors (such as the c-Met receptor)on hepatocytes, and in turn induce and promote hepatocyte proliferation.In the opposite direction, it is suggested that VEGF expressed in andsecreted from hepatocytes acts as a stimulatory factor that binds to itsreceptors (KDR and Flt-1) on sinusoidal endothelial cells, therebystimulating the proliferation and maintenance of the sinusoidalendothelial cells in the liver. Yamane et al. (1994) Oncogene9:2683-2690 observed that the endogenous expression of VEGF and VEGFreceptors (Flt and KDR) as well as HGF and c-Met are strictly regulatedin a cell-type specific manner in liver: using a flt-I cDNA as a probe,flt-1 mRNA was found to be expressed at very high levels in sinusoidalendothelial cells in normal rat liver, but was hardly detectable inhepatocytes. Similar expression pattern was found for KDR, although theexpression level was much lower. Yamane et al. further observed that, inan in vitro cell culture system, VEGF demonstrated a remarkably specificgrowth-stimulatory activity as well as maintenance activity on thesinusoidal endothelial cells.

[0027] Mochida et al. (1996) Biochem. Biophy. Res. Comm. 226:176-179conducted in vitro experiments to monitor the expression levels of VEGFand VEGFRs in isolated hepatic cells from normal livers or partiallyresected livers. They found that in 70% resected rat liver, expressionof VEGF, Flt-1 and KDR were all significantly increased. And the timingof the expression peaks for Flt-1 and KDR suggested that theupregulation of VEGFRs may be involved in proliferation of sinusoidalendothelial cells during liver regeneration.

[0028] More recently, Ajioka et al. (1999) Hepatology 29:396402 examinedthe fate of transplanted hepatic tissues in the presence of exogenousVEGF. Isolated hepatocytes of adult mice were transfected with VEGF genein vitro then transplanted intraperitoneally (i.p.) in mice, into anarea adjacent to the pancreas. The transplanted hepatocytes formed alarge number of tissue aggregates in vivo. In vitro staining showed thatthese VEGF-transfected tissues underwent substantial proliferation anddeveloped a significant vascular network therein. Thus, the resultssuggested that the expression of VEGF conferred the formation of avascular network, which in turn may promoted tissue formation. Theresults, however, showed absence of any nonparenchymal cells or growthfactors derived from them in the VEGF-transfected, transplanted hepatictissues.

[0029] Assy et al. (1999) J. Hepatol. 30:911-915 studied the effect ofVEGF as an angiogenic factor in liver regeneration following partialhepatectomy in rat. Rats undergoing 30% partial hepatectomy wereadministered intravenously (i.v.) VEGF and sacrificed at 24, 36 and 48hour postoperatively. Whilst the study showed increased DNA synthesisactivities of hepatocytes in the VEGF-treated rats at 36 and 48 h afterPHx, and suggested that stimulation of neovascularization by VEGF isimportant during liver regeneration, no statistically significantchanges in restituted liver mass were observed in VEGF-treated rats ascompared to control rats without VEGF treatment.

SUMMARY OF THE INVENTION

[0030] The present invention provides methods for promoting liver growthin a subject, comprising administering to the subject an effectiveamount of a VEGFR modulating agent. The VEGFR modulating agent usefulfor the present invention can be an agonist specific to one of the VEGFreceptors such as a Flt-1 agonist. Preferably, the Flt-1 agonist can bea Flt-1 selective VEGF variant (Flt-sel) that selectively binds toFlt-1, a growth factor that binds and activates Flt-1 such as PIGF orVEGF-B, an anti-Flt-1 agonistic antibody or a small molecule agonist. Ina preferred embodiment, the Flt-1 agonist is administered in combinationwith an angiogenic agent such as VEGF or a KDR selective variantthereof.

[0031] In another aspect, the present invention provides methods fortreating a pathological liver condition in a subject, comprisingadministering to the subject a VEGFR modulating agent in a mannereffective to alleviate the pathological liver condition. Pathologicalliver conditions that can be treated by the present invention include,but not limited to, liver failure, hepatitis, liver cirrhosis, toxicliver damage, medicamentary liver damage, hepatic encephalopathy,hepatic coma or hepatic necrosis. Preferably, the VEGFR modulating agentcomprises a Flt-1 agonist, optionally in combination with an angiogenicagent.

[0032] Also provided in the present invention are methods for protectingliver in a subject from damage due to exposure to a hepatotoxic agent,comprising administering to the subject a VEGFR modulating agent,wherein said VEGFR modulating agent effectively protects liver fromdamage. Preferably, the VEGFR modulating agent comprises a Flt-1agonist, optionally in combination with an angiogenesis agent. In oneaspect, the VEGFR modulating agent is administered prior to orconcurrent with the exposure of said subject to the hepatotoxic agent,said hepatotoxic agent being a therapeutic agent such as achemotherapeutic or radiation agent for treating cancers. As such, themethods serve to enhance the efficacy of the treatment by permitting thesubject tolerance to high doses of the therapeutic agents. In anotheraspect, the VEGFR modulating agent is administered after the exposure ofthe subject to a hepatotoxic agent but prior to any detectable liverdamage in the subject. Such methods are especially useful for treatingliver damages due to accidental exposure of the subject to a hepatotoxicagent.

[0033] In various methods of the present invention, the subject agentscan be administered to the subject through a systemic delivery system,such as a cell preparation comprising mammalian cells (e.g., CHO cells)expressing a recombinant form of the subject agent. The systemicdelivery system can comprise a slow release preparation comprisingpurified agent and a polymer matrix. Alternatively, the subject agent ofthe invention can be administered via a liver-targeted gene deliveryvector comprising a nucleic acid encoding the agent. Well establishedviral or nonviral vectors for gene therapy can be used as theliver-targeted gene delivery vector in the present invention.

[0034] An article of manufacture and a kit comprising a VEGFR modulatingagent are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIGS. 1A-1D show effects of systemic VEGF on liver mass increases.1A and 1B compare liver/brain ratios in animals implanted with CHO cellsexpressing DHFR, VEGF₁₆₅, HGF or HAg (Hakata antigen). 1C: Kidney/brainratios in animals implanted with DHFR, VEGF₁₆₅ or HGF-expressing CHOcells. 1D: Brain weights of the same groups as 1C. Error bars representstandard error of the mean.

[0036]FIGS. 2A-2D depict effects of various protein agents on culturedhepatocytes or LSECs, either alone (2A, 2B) or in a transwell setting(2C, 2D). 2A: Effects of EGF, HGF, VEGF, or VEGFR-selective agonists on3H-thymidine incorporation in primary hepatocytes when cultured alone.EGF (10 ng/ml) and HGF (Song/ml) stimulated ³H-thymidine uptake, whileVEGF, KDR^(sel), VEGF-E, Flt^(sel) and PIGF, tested at the indicatedconcentrations (ng/ml), failed to induce ³H-thymidine uptake inhepatocytes. 2B: Wild type VEGF and the KDR agonists KDR^(sel) andVEGF-E induced ³H-thymidine uptake in primary cultures of LSEC. Incontrast, the Flt-1 selective agonists, Flt^(sel) and PIGF, failed topromote LSEC proliferation. Ligands were added at the concentration of10 ng/ml, except for HGF, which was given at 50 ng/ml. 2C: In transwellLSEC/hepatocytes co-cultures, VEGF, KDR^(sel) and VEGF-E induced³H-thymidine incorporation in LSEC, whereas the Flt-1 agonists areineffective. The concentration of ligands is the same as in 2B. 2D: Intranswell LSEC/hepatocytes co-cultures, PIGF or Flt^(sel) induced³H-thymidine incorporation in primary hepatocytes to a level comparableto HGF-treated cells. In contrast, incubation with KDR^(sel) or VEGF-Eresulted in little or no stimulation of hepatocyte proliferation. Theconcentration of ligands is the same as in 2B. Error bars representstandard deviation.

[0037]FIG. 3 depicts effects of VEGF and VEGFR selective agonists on MAPkinase activation in LSEC. The ability of wild type VEGF (V) andKDR^(sel) and VEGF-E (V-E) to induce ERK activation is shown in theupper panel representing an immunoblot for phosphorylated ERK1/2.Flt^(sel) and PIGF failed to induce ERK phosphorylation althoughcomparable levels of ERK1/2 were present as indicated in the panERKimmunoblot in the lower panel. All ligands were added at 20 ng/ml.

[0038]FIG. 4 shows that Flt-1 and KDR selective agonists induceexpression of distinct and overlapping genes in LSEC. A representativeexperiment of Taqman analyses of 12 distinct gene transcripts in LSECtreated for 24 hours with 10 ng/ml VEGF, KDR^(sel) or Flt^(sel) andnormalized to control, untreated cells arbitrarily set to the value 1.Expression profiles indicate that HGF and IL-6 induction by VEGF isselectively mediated by Flt-1, whereas expression of HB-EGF or CTGF isresponsive to both Flt-1 and KDR mediated signals. TGFα and PIGF appearto be more responsive to KDR activation. See legend in upper rightcorner for bar graph coding.

[0039]FIGS. 5A and 5B depict in vivo proliferation of hepatocytes versussinusoidal endothelial cells in response to selective VEGFR activation.Quantitative analysis of proliferating hepatocytes (5A) and sinusoidalcells (5B) was performed after BrdU immunohistochemistry of liversections from animals treated with Av-LacZ, AV-KDR^(sel), orAV-Flt^(sel), 10 days after AV administration. Values are means±SEM.Level of significance was assessed by unpaired t tests; P values areindicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] General

[0041] The present invention provides, for the first time, a concertedsystem in which systemically delivered VEGFR modulating agents act in aparacrine fashion to promote liver growth. Not wishing to be bound byparticular mechanisms of action, the current system may create a localcascade of signaling events originating in sinusoidal endothelial cellsfollowing VEGF receptor activation, which is much more potent andbeneficial in promoting hepatocyte proliferation and liver growth thansystemic delivery of the principal liver mitogen, HGF. The vasculaturehas been long thought to be necessary, but not sufficient, forproliferative processes, through the delivery of nutrients and oxygenand removal of catabolic products. The present invention demonstratesthat, following an appropriate instructive signal, the vascularendothelium can be sufficient to initiate and amplify a growth/survivalprocess that may overcome the set point for final organ size and protectthe parenchyma from injury.

[0042] Particularly enticing are the surprising findings on a novelfunction of the VEGFR-1 (Flt-1) RTK in regulating key paracrineactivities of LSEC, which in turn leads to liver proliferation andprotection. Strikingly, in spite of the lack of stimulation ofangiogenesis, activation of VEGFR-1 was sufficient to substantiallyprotect the liver parenchyma from toxic injury. Indeed, the presentinvention provides the first evidence that protective effects onparenchymal cells mediated by the endothelium can be uncoupled fromstimulation of angiogenesis.

[0043] Given that the known dose-limiting effects of VEGF (e.g.hypotension, edema) (Yang et al. (1998) J. Pharmacol. Exp. Ther.284:103-10) are associated with KDR activation (Kliche and Waltenberger(2001) IUBMB Life 52:61-6), it is contemplated that a Flt-1 agonist,such as a Flt-selective VEGF variant, can form the basis of atherapeutic scheme aimed toward liver protection. The addition of a KDRagonist or other angiogenic factor at a lower ratio may result in amaximal therapeutic benefit, by providing stimulation of angiogenesis.Alternatively, a VEGF variant that preferentially activates Flt-1 versusKDR might combine optimal characteristics of safety and efficacy. Thepotential indications include acute liver damage induced by variousdrugs., chemotherapy, or toxins as well as chronic injury, includingcirrhosis.

[0044] Compositions of the invention and Their Productions

[0045] The present invention relates to uses of various agents capableof modulating VEGFR activities in the liver. The term “VEGF receptor” or“VEGFR” as used herein refers to a cellular receptor for VEGF,ordinarily a cell-surface receptor found on vascular endothelial cells,as well as fragments and variants thereof which retain the ability tobind VEGF (such as fragments or truncated forms of the extracellulardomain). Some examples of VEGFR include the protein kinase receptorsreferred to in the literature as Flt-1 and KDR/Flk-1. DeVries et al.Science, 255:989 (1992); Shibuya et al. Oncogene, 5:519 (1990); Matthewset al. Proc. Nat. Acad. Sci., 88:9026 (1991); Terman et al. Oncogene,6:1677 (1991); and Terman et al. Biochem. Biophys. Res. Commun.,187:1579 (1992). The Flt-1 (fms-like-tyrosine kinase) and KDR (kinasedomain region) receptors bind VEGF with high affinity. Flk-1 (fetalliver kinase-1), the murine homolog of KDR, shares 85% sequence identitywith human KDR. Ferrara (1999) Kidney Intl. 56:794-814. Both Flt-1 andKDR/Flk-1 have seven immunoglobulin (Ig)-like domains in theextracellular domain (ECD), a single transmembrane region and aconsensus tyrosine kinase (TK) sequence, which is interrupted by akinase-insert domain. Flt-1 has the highest affinity for rhVEGF₁₆₅, witha Kd of approximately 10 to 20 pM. KDR has a lower affinity for VEGF,with a Kd of approximately 75 to 125 pM.

[0046] Other VEGF receptors include those that can be cross-link labeledwith VEGF, or that can be co-immunoprecipitated with KDR or Fit-1. Anadditional VEGF receptor that binds VEGF₁₆₅ but not VEGF₁₂₁ has beenidentified. Soker et al (1998) Cell 92:735-45. The isoform-specific VEGFbinding site is identical to human neuropilin-1, a receptor for thecollapsin/semaphorin family that mediates neuronal cell guidance.

[0047] The Flt-1 and KDR receptors mainly exist as a bound receptor onthe surface of vascular endothelial cells, although they can also bepresent in non-endothelial cells. Some soluble forms of VEGFR have alsobeen found. For example, a cDNA coding an alternatively spliced solubleform of Flt-1 (sFlt-1), lacking the seventh Ig-like domain,transmembrane sequence, and the cytoplasmic domain, has been identifiedin human umbilical vein endothelial cells (HUVECs). Kendall et al.(1996) Biochem. Biophys. Res. Comm. 226:324-328.

[0048] The term “agent” or, alternatively, “compound” as used hereinrefers broadly to any substance with identifiable molecular structureand physiochemical property. Non-limiting examples of agents capable ofmodulating VEGFR activities include antibodies, proteins, peptides,glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics,pharmacological agents and their metabolites, transcriptional andtranslation control sequences, and the like.

[0049] The VEGFR modulating agents encompassed by the invention can beeither an agonist or an antagonist of a VEGFR. An “agonist” is an agentthat mediates or activate the biological activity of its target. Forexample, a VEGFR agonist can be a growth factor ligand or an antibodythat binds to the VEGFR's extracellular domain and triggers its signaltransduction activity. Alternatively, a VEGFR agonist can be a smallmolecule compound that binds to the VEGFR's cytoplasmic domain andmediates its tyrosine phosphorylation. An “antagonist”, on the otherhand, is one which blocks, inhibits or reduces biological activity ofits target. Such inhibition can occur by any means, e.g. by interferingwith: ligand binding to the receptor, receptor complex formation,tyrosine kinase activity of a tyrosine kinase receptor in a receptorcomplex and/or phosphorylation of tyrosine kinase residue(s) in or bythe receptor.

[0050] In a preferred embodiment, the agonist or antagonist of theinvention is “selective” or “specific” to Flt-1, i.e., it exclusively orpreferably modulates Flt-1 over other receptor tyrosine kinases such asKDR. In another embodiment, the agonist or antagonist of the inventionis “selective” or “specific” to KDR, i.e., it exclusively or preferablymodulates KDR over other receptor tyrosine kinases such as Flt-1.

[0051] In one aspect, the VEGFR agonist of the invention comprises aVEGF variant polypeptide capable of selectively binding to Flt-1(referred hereinafter as “Flt-1 selective VEGF variant”, or “Flt-sel”,or “Flt^(sel)”). The term “VEGF” as used herein refers to the 165-aminoacid vascular endothelial cell growth factor and related 121-, 189-, and206-amino acid vascular endothelial cell growth factors, as described byLeung et al. Science, 246:1306 (1989), and Houck et al. Mol. Endocrin.,5:1806 (1991), together with the naturally occurring allelic andprocessed forms thereof. The term “VEGF” is also used to refer totruncated forms of the polypeptide comprising amino acids 8 to 109 or 1to 109 of the 165-amino acid human vascular endothelial cell growthfactor. Reference to any such forms of VEGF may be identified in thepresent application, e.g., by “VEGF (8-109),” “VEGF (1-109)” or“VEGF₁₆₅.” The amino acid positions for a “truncated” native VEGF arenumbered as indicated in the native VEGF sequence. For example, aminoacid position 17 (methionine) in truncated native VEGF is also position17 (methionine) in native VEGF. The truncated native VEGF has bindingaffinity for the KDR and Flt-1 receptors comparable to native VEGF.

[0052] The term “VEGF variant” as used herein refers to a VEGFpolypeptide which includes one or more amino acid mutations in thenative VEGF sequence. Optionally, the one or more amino acid mutationsinclude amino acid substitution(s). For purposes of shorthanddesignation of VEGF variants described herein, it is noted that numbersrefer to the amino acid residue position along the amino acid sequenceof the putative native VEGF (provided in Leung et al., supra and Houcket al., supra.).

[0053] VEGF and variants thereof for use in the present invention can beprepared by a variety of methods well known in the art. Preferably, theVEGF employed in the methods of the present invention comprisesrecombinant VEGF₁₆₅. Amino acid sequence variants of VEGF can beprepared by mutations in the VEGF DNA. Such variants include, forexample, deletions from, insertions into or substitutions of residueswithin the amino acid sequence shown in Leung et al., supra and Houck etal., supra. Any combination of deletion, insertion, and substitution maybe made to arrive at the final construct having the desired activity.Obviously, the mutations that will be made in the DNA encoding thevariant must not place the sequence out of reading frame and preferablywill not create complementary regions that could produce secondary mRNAstructure. EP 75,444A.

[0054] The VEGF variants optionally are prepared by site-directedmutagenesis of nucleotides in the DNA encoding the native VEGF or phagedisplay techniques, thereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture.

[0055] While the site for introducing an amino acid sequence variationis predetermined, the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed VEGF variants screened for the optimal combination ofdesired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well-known, suchas, for example, site-specific mutagenesis.

[0056] Preparation of the VEGF variants described herein is preferablyachieved by phage display techniques, such as those described in the PCTpublication WO 00/63380.

[0057] After such a clone is selected, the mutated protein region may beremoved and placed in an appropriate vector for protein production,generally an expression vector of the type that may be employed fortransformation of an appropriate host.

[0058] Amino acid sequence deletions generally range from about 1 to 30residues, more preferably 1 to 10 residues, and typically arecontiguous.

[0059] Amino acid sequence insertions include amino- and/orcarboxyl-terminal fusions of from one residue to polypeptides ofessentially unrestricted length as well as intrasequence insertions ofsingle or multiple amino acid residues. Intrasequence insertions (i.e.,insertions within the native VEGF sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5. An example of a terminalinsertion includes a fusion of a signal sequence, whether heterologousor homologous to the host cell, to the N-terminus to facilitate thesecretion from recombinant hosts.

[0060] Additional VEGF variants are those in which at least one aminoacid residue in the native VEGF has been removed and a different residueinserted in its place. Such substitutions may be made in accordance withthose shown in Table 1. TABLE 1 Original Residue Exemplary SubstitutionsAla (A) gly; ser Arg (R) lys Asn (N) gln; his Asp (D) glu Cys (C) serGln (Q) asn Glu (E) asp Gly (G) ala; pro His (H) asn; gln Ile (I) leu;val Leu (L) ile; val Lys (K) arg; gln; glu Met (M) leu; tyr; ile Phe (F)met; leu; tyr Ser (S) thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val(V) ile; leu

[0061] Changes in function or immunological identity may be made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain. The substitutions thatin general are expected to produce the greatest changes in the VEGFvariant properties will be those in which (a) glycine and/or proline (P)is substituted by another amino acid or is deleted or inserted; (b) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, oralanyl; (c) a cysteine residue is substituted for (or by) any otherresidue; (d) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) a residue havingan electronegative charge, e.g., glutamyl or aspartyl; (e) a residuehaving an electronegative side chain is substituted for (or by) aresidue having an electropositive charge; or (f) a residue having abulky side chain, e.g., phenylalanine, is substituted for (or by) onenot having such a side chain, e.g., glycine.

[0062] The effect of the substitution, deletion, or insertion may beevaluated readily by one skilled in the art using routine screeningassays. For example, a phage display-selected VEGF variant may beexpressed in recombinant cell culture, and, optionally, purified fromthe cell culture. The VEGF variant may then be evaluated for KDR orFlt-1 receptor binding affinity and other biological activities, such asthose disclosed in the present application. The binding properties oractivities of the cell lysate or purified VEGF variant can be screenedin a suitable screening assay for a desirable characteristic. Forexample, a change in the immunological character of the VEGF variant ascompared to native VEGF, such as affinity for a given antibody, may bedesirable. Such a change may be measured by a competitive-typeimmunoassay, which can be conducted in accordance with techniques knownin the art. The respective receptor binding affinity of the VEGF variantmay be determined by ELISA, RIA, and/or BLAcore assays, known in the artand described further in the Examples below. Preferred VEGF variants ofthe invention will also exhibit activity in KIRA assays (such asdescribed in the Examples) reflective of the capability to inducephosphorylation of the KDR receptor. Preferred VEGF variants of theinvention will additionally or alternatively induce endothelial cellproliferation (which can be determined by known art methods such as theHUVEC proliferation assay in the Examples). In addition to the specificVEGF variants disclosed herein, the VEGF variants described in Keyt etal. (1996) J. Biol. Chem. 271:5638-5646 are also contemplated for use inthe present invention.

[0063] Flt-sel and methods of making the same have been known and aredescribed in the Example sections below. Additional disclosures relatingto Flt-sel can be found in, for example, the PCT publication WO 00/63380and Li et al. (2000) J. Biol. Chem. 275:29823-29828. Preferred Flt-selvariants include one or more amino acid mutations and exhibit bindingaffinity to the Flt-1 receptor which is equal to or greater (≧) than thebinding affinity of native VEGF to the Flt-1 receptor, and even morepreferably, such VEGF variants exhibit less binding affinity (<) to theKDR receptor than the binding affinity exhibited by native VEGF to KDR.When binding affinity of such VEGF variant to the Flt-1 receptor isapproximately equal (unchanged) or greater than (increased) as comparedto native VEGF, and the binding affinity of the VEGF variant to the KDRreceptor is less than or nearly eliminated as compared to native VEGF,the binding affinity of the VEGF variant, for purposes herein, isconsidered “selective” for the Flt-1 receptor. Preferred Flt-1 selectiveVEGF variants of the invention will have at least 10-fold less bindingaffinity to KDR receptor (as compared to native VEGF), and even morepreferably, will have at least 100-fold less binding affinity to KDRreceptor (as compared to native VEGF). The respective binding affinityof the VEGF variant may be determined by ELISA, RIA, and/or BIAcoreassays, known in the art and described in the PCT publication WO00/63380.

[0064] In some aspects of the invention, various methods for livertreatment further comprise administering an agent capable of modulatingKDR activities. For example, a KDR agonist can be administered incombination with a Flt-1 agonist to promote liver growth or liverregeneration. KDR has been identified as the major receptor tyrosinekinase that mediates VEGF's activities in endothelial cellproliferation. Thus, agonists of KDR and Flt-1 will induce concertedproliferation of both the SECs and hepatocytes, thereby promoting acoordinated growth of liver.

[0065] In one aspect, the KDR agonist comprises a VEGF variantpolypeptide capable of selectively binding to KDR (referred hereinafteras “KDR selective VEGF variant”, or “KDR-sel”, or “KDR^(sel)”). KDR-selVEGF variants and methods of making the same are described in detail inthe Example sections below. Additional disclosures relating to KDR-selcan be found in, for example, the PCT publication WO 00/63380 and Li etal. (2000) J. Biol. Chem. 275:29823-29828. Preferred KDR-sel include oneor more amino acid mutations and exhibit binding affinity to the KDRreceptor which is equal to or greater (≧) than the binding affinity ofnative VEGF to the KDR receptor, and even more preferably, the VEGFvariants exhibit less binding affinity (<) to the flt-1 receptor thanthe binding affinity exhibited by native VEGF to Flt-1. When bindingaffinity of such VEGF variant to the KDR receptor is approximately equal(unchanged) or greater than (increased) as compared to native VEGF, andthe binding affinity of the VEGF variant to the flt-1 receptor is lessthan or nearly eliminated as compared to native VEGF, the bindingaffinity of the VEGF variant, for purposes herein, is considered“selective” for the KDR receptor. Preferred KDR-sel of the inventionwill have at least 10-fold less binding affinity to Flt-1 receptor (ascompared to native VEGF), and even more preferably, will have at least100-fold less binding affinity to Flt-1 receptor (as compared to nativeVEGF). The respective binding affinity of the VEGF variant may bedetermined by ELISA, RIA, and/or BIAcore assays that are known in theart. Preferred KDR-sel of the invention will also exhibit activity inKIRA assays reflective of the capability to induce phosphorylation ofthe KDR receptor. Preferred KDR selective VEGF variants of the inventionwill additionally or alternatively induce endothelial cell proliferation(which can be determined by known methods such as the HUVECproliferation assay).

[0066] In one aspect, the VEGFR modulating agents of the invention, suchas VEGF and variants thereof, are produced by recombinant methods.Isolated DNA used in these methods is understood herein to meanchemically synthesized DNA, cDNA, chromosomal, or extrachromosomal DNAwith or without the 3′- and/or 5′-flanking regions. Preferably, the VEGFand variants thereof herein are made by synthesis in recombinant cellculture.

[0067] For such synthesis, it is first necessary to secure nucleic acidthat encodes a VEGF or VEGF variant. DNA encoding a VEGF molecule may beobtained from bovine pituitary follicular cells by (a) preparing a cDNAlibrary from these cells, (b) conducting hybridization analysis withlabeled DNA encoding the VEGF or fragments thereof (up to or more than100 base pairs in length) to detect clones in the library containinghomologous sequences, and (c) analyzing the clones by restriction enzymeanalysis and nucleic acid sequencing to identify full-length clones. Iffull-length clones are not present in a cDNA library, then appropriatefragments may be recovered from the various clones using the nucleicacid sequence information disclosed herein for the first time andligated at restriction sites common to the clones to assemble afull-length clone encoding the VEGF. Alternatively, genomic librarieswill provide the desired DNA.

[0068] Once this DNA has been identified and isolated from the library,it is ligated into a replicable vector for further cloning or forexpression.

[0069] In one example of a recombinant expression system, aVEGF-encoding gene is expressed in a cell system by transformation withan expression vector comprising DNA encoding the VEGF. It is preferableto transform host cells capable of accomplishing such processing so asto obtain the VEGF in the culture medium or periplasm of the host cell,i.e., obtain a secreted molecule.

[0070] “Transfection” refers to the taking up of an expression vector bya host cell whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan, for example, CaPO₄ and electroporation. Successful transfectionis generally recognized when any indication of the operation of thisvector occurs within the host cell.

[0071] “Transformation” refers to introducing DNA into an organism sothat the DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described by Cohen, Proc. Natl.Acad. Sci. (USA), 69: 2110 (1972) and Mandel et al. J. Mol. Biol., 53:154 (1970), is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham and vander Eb, Virology, 52: 456-457 (1978), is preferred. General aspects ofmammalian cell host system transformations have been described by Axelin U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. Transformations intoyeast are typically carried out according to the method of Van Solingenet al. J. Bact., 130: 946 (1977) and Hsiao et al. Proc. Natl. Acad. Sci.(USA), 76: 3829 (1979). However, other methods for introducing DNA intocells such as by nuclear injection or by protoplast fusion may also beused.

[0072] The vectors and methods disclosed herein are suitable for use inhost cells over a wide range of prokaryotic and eukaryotic organisms.

[0073] In general, of course, prokaryotes are preferred for the initialcloning of DNA sequences and construction of the vectors useful in theinvention. For example, E. coli K12 strain MM 294 (ATCC No. 31,446) isparticularly useful. Other microbial strains that may be used include E.coli strains such as E. coli B and E. coli X1776 (ATCC No. 31,537).These examples are, of course, intended to be illustrative rather thanlimiting.

[0074] Prokaryotes may also be used for expression. The aforementionedstrains, as well as E. coli strains W3110 (F—, lambda-, prototrophic,ATCC No. 27,325), K5772 (ATCC No. 53,635), and SR101, bacilli such asBacillus subtilis, and other enterobacteriaceae such as Salmonellatyphimurium or Serratia marcesans, and various pseudomonas species, maybe used.

[0075] In general, plasmid vectors containing replicon and controlsequences that are derived from species compatible with the host cellare used in connection with these hosts. The vector ordinarily carries areplication site as well as marking sequences that are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species (see, e.g., Bolivar et al. Gene, 2:95 (1977). pBR322contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other microbial plasmid or phage, must also contain, or bemodified to contain, promoters that can be used by the microbialorganism for expression of its own proteins.

[0076] Those promoters most commonly used in recombinant DNAconstruction include the β-lactamase (penicillinase) and lactosepromoter systems Chang et al. Nature, 375:6115 (1978); Itakura et al.Science, 198:1056 (1977); Goeddel et al. Nature, 281:544 (1979)) and atryptophan (trp) promoter system (Goeddel et al. Nucleic Acids Res.,8:4057 (1980); EPO Appi. Publ. No. 0036,776). While these are the mostcommonly used, other microbial promoters have been discovered andutilized, and details concerning their nucleotide sequences have beenpublished, enabling a skilled worker to ligate them functionally withplasmid vectors (see, e.g., Siebenlist et al. Cell, 20:269 (1980)).

[0077] In addition to prokaryotes, eukaryotic microbes, such as yeastcultures, may also be used. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among eukaryotic microorganisms,although a number of other strains are commonly available. Forexpression in Saccharomyces, the plasmid YRp7, for example (Stinchcombet al. Nature, 282:39 (1979); Kingsman et al. Gene, 7:141 (1979);Tschemper et al. Gene, 10:157 (1980)), is commonly used. This plasmidalready contains the trp1 gene that provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample, ATCC No. 44,076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). Thepresence of the trp1 lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

[0078] Suitable promoting sequences in yeast vectors include thenromoters for 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem.,255:2073 (1980)) or other glycolytic enzymes (Hess et al. J. Adv. EnzymeReg., 7:149 (1968); Holland et al. Biochemistry, 17:4900 (1978)), suchas enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructo-kinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. In constructingsuitable expression plasmids, the termination sequences associated withthese genes are also ligated into the expression vector 3′ of thesequence desired to be expressed to provide polyadenylation of the mRNAand termination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

[0079] In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)). Examples of such useful host cell linesare VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, andW138, BHK, COS-7, 293, and MDCK cell lines. Expression vectors for suchcells ordinarily include (if necessary) an origin of replication, apromoter located in front of the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsites, and transcriptional terminator sequences.

[0080] For use in mammalian cells, the control functions on theexpression vectors are often provided by viral material. For example,commonly used promoters are derived from polyoma, Adenovirus2, and mostfrequently Simian Virus 40 (SV40). The early and late promoters of SV40virus are particularly useful because both are obtained easily from thevirus as a fragment that also contains the SV40 viral origin ofreplication (Fiers et al. Nature, 273:113 (1978)). Smaller or largerSV40 fragments may also be used, provided there is included theapproximately 250-bp sequence extending from the HindIII site toward theBglI site located in the viral origin of replication. Further, it isalso possible, and often desirable, to utilize promoter or controlsequences normally associated with the desired gene sequence, providedsuch control sequences are compatible with the host cell systems.

[0081] An origin of replication may be provided either by constructionof the vector to include an exogenous origin, such as may be derivedfrom SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or maybe provided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

[0082] Satisfactory amounts of protein are produced by cell cultures;however, refinements, using a secondary coding sequence, serve toenhance production levels even further. One secondary coding sequencecomprises dihydrofolate reductase (DHFR) that is affected by anexternally controlled parameter, such as methotrexate (MTX), thuspermitting control of expression by control of the methotrexateconcentration.

[0083] In selecting a preferred host cell for transfection by thevectors of the invention that comprise DNA sequences encoding both VEGFand DHFR protein, it is appropriate to select the host according to thetype of DHFR protein employed. If wild-type DHFR protein is employed, itis preferable to select a host cell that is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium that lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, Proc. Natl.Acad. Sci. (USA), 77:4216 (1980).

[0084] On the other hand, if DHFR protein with low binding affinity forMTX is used as the controlling sequence, it is not necessary to useDHFR-deficient cells. Because the mutant DHFR is resistant tomethotrexate, MTX-containing media can be used as a means of selectionprovided that the host cells are themselves methotrexate sensitive. Mosteukaryotic cells that are capable of absorbing MTX appear to bemethotrexate sensitive. One such useful cell line is a CHO line, CHO-K1(ATCC No. CCL 61).

[0085] Construction of suitable vectors containing the desired codingand control sequences employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to prepare the plasmids required.

[0086] If blunt ends are required, the preparation may be treated for 15minutes at 15° C. with 10 units of Polymerase I (Klenow),phenol-chloroform extracted, and ethanol precipitated.

[0087] Size separation of the cleaved fragments may be performed using,by way of example, 6 percent polyacrylamide gel described by Goeddel etal. Nucleic Acids Res., 8:4057 (1980).

[0088] To confirm correct sequences were constructed in plasmids, theligation mixtures are typically used to transform E. coli K12 strain 294(ATCC 31,446) or other suitable E. coli strains, and successfultransformants selected by ampicillin or tetracycline resistance whereappropriate. Plasmids from the transformants are prepared and analyzedby restriction mapping and/or DNA sequencing by the method of Messinget. al. Nucleic Acids Res., 9:309 (1981) or by the method of Maxam etal. Methods of Enzymology, 65:499 (1980).

[0089] After introduction of the DNA into the mammalian cell host andselection in medium for stable transfectants, amplification ofDHFR-protein-coding sequences is effected by growing host cell culturesin the presence of approximately 20,000-500,000 nM concentrations ofmethotrexate (MTX), a competitive inhibitor of DHFR activity. Theeffective range of concentration is highly dependent, of course, uponthe nature of the DHFR gene and the characteristics of the host.Clearly, generally defined upper and lower limits cannot be ascertained.Suitable concentrations of other folic acid analogs or other compoundsthat inhibit DHFR could also be used. MIX itself is, however,convenient, readily available, and effective.

[0090] In some aspects of the invention, the Flt-1 agonist comprises agrowth factor that selectively binds to and activates Flt-1. Severalnaturally occurring VEGF homologues that specifically bind to Flt-1 butnot KDR have been identified, including without limiting to, placentalgrowth factor (PIGF) and VEGF-B. PIGF has an amino acid sequence thatshares 53% identity with the platelet-derived growth factor-like domainof VEGF. Park et al. (1994) J. Biol. Chem. 269:25646-54; Maglione et al.(1993) Oncogene 8:925-31. As with VEGF, different species of PIGF arisefrom alternative splicing of mRNA, and the protein exists in dimericform. Park et al., supra. Both PIGF-1 and PIGF-2 bind to Flt-1 with highaffinity, but neither is able to interact with KDR. Park et al., supra.

[0091] VEGF-B is produced as two isoforms (167 and 185 residues) thatalso appear to specifically bind Flt-1. Pepper et al. (1998) Proc. Natl.Acad. Sci. USA 95:11709-11714. Similar to the long forms of VEGF, VEGF-Bis expressed as a membrane-bound protein that can be released in asoluble form after addition of heparin. VEGF-B and VEGF are also able toform heterodimers, when coexpressed. Olofsson et al. (1996) Proc. Natl.Acad. Sci. USA 93:2576-2581.

[0092] Compounds useful in the invention include small oraganicmolecules that exert their modulating functions at the intracellulartyrosine kinase domain of the RTKs. In certain preferred embodiments,small molecule agonists are employed to stimulate tyrosinephosphorylation, thereby activating the corresponding signaling pathway.In other embodiments, small molecule inhibitors or antagonists are usedto block and/or deactivate the RTK activities. Many small moleculecompounds can be used for the purpose of this invention. These include,but not limited to, bis monocyclic, bicyclic or heterocyclic arylcompounds, vinylene-azaindole derivatives (PCT WO 94/14808) and1-cycloproppyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992), styrylcompounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EPApplication No. 0 566 266 A1), selenoindoles and selenides (PCT WO94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) andbenzylphosphonic acid compounds (PCT WO 91/15495).

[0093] Compounds useful in the present invention include agonist orantagonist antibodies. Antibodies of the present invention can be eitherspecific against a receptor (such as Flt-1), or specific against aligand of the receptor, so long as they exert the necessary agonistic orantagonistic activity. Preferred antibodies of the invention includeanti-Flt-1 antibodies. More preferably, the anti-Flt-1 antibodyselectively binds to and modulate Flt-1, without affecting the KDRfunction.

[0094] The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length or intact monoclonalantibodies), polyclonal antibodies, multivalent antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments so long as they exhibit the desired biological activity. It isalso contemplated that non-human antibodies, chimeric antibodies,humanized antibodies or human antibodies can be used for the purpose ofthe invention. Methods of preparing various antibodies suitable for theinvention are known to the skilled artisan.

[0095] A naturally occurring antibody comprises four polypeptide chains,two identical heavy (H) chains and two identical light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (V_(H)) and a heavy chain constant region,which in its native form is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(V_(L)) and a light chain constant region. The light chain constantregion is comprised of one domain, C_(L). The V_(H) and V_(L) regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The light chains of antibodies from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa (κ) and lambda (λ), based on the amino acid sequences oftheir constant domains. Depending on the amino acid sequences of theconstant domains of their heavy chains, antibodies (immunoglobulins) canbe assigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgA-1,IgA-2, and etc. The heavy chain constant domains that correspond to thedifferent classes of immunoglobulins are called α, δ, ε, γ, and μ,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well knownand described generally in, for example, Abbas et al. Cellular and Mol.Immunology, 4th ed. (2000). An antibody may be part of a larger fusionmolecule, formed by covalent or noncovalent association of the antibodyor antibody portion with one or more other proteins or peptides.Examples of such fusion proteins include use of the streptavidin coreregion to make a tetrameric scFv molecule (Kipriyanov et al. (1995)Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue,a marker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058).

[0096] Other agents capable of modulating Flt-1 or KDR activitiesinclude, for example but not limited to, soluble extracellular domainpeptides of Flt-1 or KDR, Flt-1 or KDR binding peptides, Flt-1 or KDRspecific ribozymes, antisense polynucleotides and RNA ligands. Forexample, soluble Flt-1 extracellular fragments as antagonists aredescribed in U.S. Pat. No. 6,100,071.

[0097] Assay Methods of the Invention

[0098] In one aspect, the invention provides methods of using VEGFRagonists to upregulate gene expressions of factors that are important inregulating liver activities. In a preferred embodiment, the expressionof HGF in nonparenchymal cells is upregulated. Methods and techniquesfor detecting levels of mRNA expression or protein expression in targetcells/tissues are known to those skill in the art. For example, the HGFgene expression level can be detected by known nucleic acidhybridization assays, using probes capable of hybridizing to HGFpolynucleotides, under conditions suitable for the hybridization andsubsequent detection and measurement. Methods useful for detecting HGFgene expression include but not limited to southern hybridization(Southern (1975) J. Mol. Biol. 98:503-517), northern hybridization (see,e.g., Freeman et al. (1983) Proc. Natl. Acad. Sci. USA 80:4094-4098),restriction endonuclease mapping (Sambrook et al. (1989) MolecularCloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor LaboratoryPress, New York), RNase protection assays (Current Protocols inMolecular Biology, John Wiley and Sons, New York, 1997), DNA sequenceanalysis, and polymerase chain reaction amplification (PCR; U.S. Pat.Nos. 4,683,202, 4,683,195, and 4,889,818; Gyllenstein et al. 1988, ProcNatl. Acad. Sci. USA 85:7652-7657; Ochman et al. 1988, Genetics120:621-623; Loh et al. 1989, Science 243:217-220) followed by Southernhybridization with probes specific for the HGF gene, in various celltypes. Other methods of amplification commonly known in the art can beemployed. The stringency of the hybridization conditions for northern orSouthern blot analysis can be manipulated to ensure detection of nucleicacids with the desired degree of relatedness to the specific probesused. The expression of HGF in a cell or tissue sample can also bedetected and quantified using in situ hybridization techniques accordingto, for example, Current Protocols in Molecular Biology, John Wiley andSons, New York, 1997.

[0099] The HGF protein levels can be detected by immunoassays usingantibodies specific to HGF. Various immunoassays known in the art can beused, including but not limited to competitive and non-competitive assaysystems using techniques such as radioimmunoassay, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitin reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels),western blot analysis, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hernagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays,immunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

[0100] The invention provides methods for promoting liver growth andhepatocvte cell proliferation by administering an effective amount ofVEGFR agonists. The promoting effects of the present invention can beassessed either in vitro or in vivo, using methods known in the art.Drakes et al. (1997) J. Immunol. 159:4268; Omori et al. (1997)Hepatology 26:720; U.S. Pat. No. 5,227,158.

[0101] In one embodiment of the invention, hepatocytes and othernonparenchymal liver cells are isolated from the target livers andresuspended in appropriate tissue culture medium to induce celladherence. If necessary, different cell fractions can be furtherseparated (e.g., parenchymal cells from nonparenchymal cells) bycentrifugation at different speeds for different length of time. Cellproliferation is assessed during culture using methods known in the art,including but not limited to, measuring the rate of DNA synthesis (see,e.g., Nakamura et al. (1984) supra), trypan blue dyeexclusion/hemacytometer counting (see, e.g., Omiri et al. (1997) supra),or flow cytometry (see, e.g., Drakes (1997) supra).

[0102] In another embodiment, the proliferative effect of a VEGFRagonist on hepatic cells and liver organ as a whole is measured in vivousing, for example, histochemistry assays of the liver tissue samples.In a preferred aspect, in vivo proliferation of hepatic cells isassessed by reactivity to an antibody directed against a protein knownto be present in higher concentrations in proliferating cells than innon-proliferating cells, such as proliferating cell nuclear antigen(PCNA or cyclin). Rodgers et al. (1997) J. Burn Care Rehabil.18:381-388. A more preferred method is the BrdU immunohistochemistryassay as previously described by Gerber et al. (1999) Development126:1149-1159.

[0103] Treatment of Pathological Liver Conditions

[0104] According to one embodiment, the invention provides methods fortreating a pathological liver condition in a subject. As used herein,“treatment” refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.

[0105] An “effective amount” refers to an amount effective, at dosagesand for periods of time necessary, to achieve the desired therapeutic orprophylactic result. A “therapeutically effective amount” of theantibody may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the antibody toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

[0106] The phrase “pathological liver condition” is used interchangeablywith “liver disorder” or “liver disease” to indicate any structuraland/or functional liver abnormalities. Non-limiting examples ofpathological liver condition include those conditions associated withliver failure, hepatitis (acute, chronic or alcohol), liver cirrhosis,toxic liver damage, medicamentary liver damage, hepatic encephalopathy,hepatic coma or hepatic necrosis.

[0107] Protection Against Liver Damage

[0108] In one aspect, the invention provides methods for protectingliver from damage in a subject susceptible to conditions or factorscausative of liver damage. The phrase “liver damage” is used herein inthe broadest sense, and indicates any structural or functional liverinjury resulting, directly or indirectly, from internal or externalfactors or their combinations. Liver damage can be induced by a numberof factors including, but not limited to, exposure to hepatotoxiccompounds, radiation exposure, mechanical liver injuries, geneticpredisposition, viral infections, autoimmune disease, such as,autoimmune chronic hepatitis and as a result of elevated in vivo levelsof proteins, such as activin and TGF-.beta.

[0109] Liver damage induced by hepatotoxic compounds includes directcytotoxicity including drug hypersensitivity reactions, cholestasis, andinjury to the vascular endothelium.

[0110] A number of hepatotoxic compounds, including certaintherapeutics, induce cytotoxicity. Hepatotoxic compounds can produceliver cytotoxicity by direct chemical attack or by the production of atoxic metabolite. Although the exact mechanism of hepatotoxicity isuncertain, the products of reductive metabolism are highly reactivespecies that bind to cellular macromolecules and cause lipidperoxidation and inactivation of drug metabolizing and other enzymes.The membrane injury provokes release of calcium from mitochondria andsmooth endoplasmic reticulum and appears to interfere with the calciumion pump, which normally prevents cytosolic accumulation of calcium. Thedeleterious effect on cell metabolism with resultant calciumaccumulation, the loss of potassium and enzymes from the cytoplasm, andthe loss of essential energy that results from mitochondrial injury allcontribute to the necrosis of hepatic tissue.

[0111] Many hepatotoxic compounds unpredictably produce liver damage ina small proportion of recipients. In some patients, the liver damage isreferred to as a hypersensitivity reaction and is like that of a drugreaction, where the patient presents with fever, rash and eosinophiliaand has a recurrence of symptoms upon rechallenge of the drug. In othersituations, the mechanism for injury is unknown and may representaberrant metabolism in susceptible patients that permits the productionor accumulation of hepatotoxic metabolites.

[0112] Those drugs inducing cytotoxicity by direct chemical attackinclude the following: Anesthetics, such as Enflurane, Fluroxene,Halothane, and Methoxyflurane; Neuropsychotropics, such as, Cocaine,Hydrazides, Methylphenidate, and Tricyclics; Anticonvulsants, such as,Phenyloin and Valproic acid; Analgesics, such as, Acetaminophen,Chlorzoxazone, Dantrolene, Diclofenac, Ibuprofen, Indomethacin,Salicylates, Tolmetin, and Zoxazolamine; Hormones, such as,Acetohexamide, Carbutamide, Glipizide, Metahexamide, Propylthiouracil,Tamoxifen, Diethylstilbestrol; Antimicrobials, such as, Amphotericin B,Clindamycin, Ketoconazole, Mebendazole, Metronidazole, Oxacillin,Paraminosalicylic acid, Penicillin, Rifampicin, Sulfonamides,Tetracycline, and Zidovudine; Cardiovascular drugs, such as, Amiodarone,Dilitiazem, a-Methyldopa, Mexiletine, Hydrazaline, Nicotinic acid,Papaverine, Perhexiline, Procainamide, Quinidine, and Tocainamide; andImmunosuppressives and Antineoplastics, such as, Asparaginase,Cisplatin, Cyclophosphamide, Dacarbazine, Doxorubicin, Fluorouracil,Methotrexate, Mithramycin, 6-MP, Nitrosoureas, Tamoxifen, Thioguanine,and Vincristine; and Miscellaneous drugs, such as, Disulfiram, Iodideion, Oxyphenisatin, Vitamin A and Paraminobenzoic acid.

[0113] Those hepatotoxic compounds producing hypersensitivity reactionin the liver include the following: Phenyloin, Paramino salicylic acid,Chlorpromazine, Sulfonamides, Erythromycin estolate, Isoniazid,Halothane, Methyldopa, and Valproic acid.

[0114] Hepatotoxic compounds including cholestasis, an arrest in theflow of bile, may take several forms. Centribular cholestasis isaccompanied by portal inflammatory changes. Bile duct changes have beenreported with some drugs such as erythromycin, while pure canalicularcholestasis is characteristic of other drugs such as the anabolicsteroids. Chronic cholestasis has been linked to such drugs asmethyltestosterone and estradiol.

[0115] Those hepatotoxic compounds inducing cholestatic disease includethe following: Contraceptive steroids, androgenic steroids, anabolicsteroids, Acetylsalicylic acid, Azathioprine, Benzodiazepine,Chenodeoxycholic acid, Chlordiazepoxide, Erythromycin estolate,Fluphenazine, Furosemide, Griseofulvin, Haloperidol, Imipramine,6-Mercaptopurine, Methimazole, Methotrexate, Methyldopa,Methylenediamine, Methyltestosterone, Naproxen, Nitrofurantoin,Penicillamine, Perphenazine, Prochlorperazine, Promazine, Thiobendazole,Thioridazine, Tolbutamide, Trimethoprimsulfamethoxazole, Arsenic,Copper, and Paraquat.

[0116] Some drugs, although primarily cholestatic, can also producehepatoxicity, and therefore the liver injury they cause is mixed. Thedrugs causing mixed liver injury include, for example, the following:Chlorpromazine, Phenylbutazone, Halothane, Chlordiazepoxide, Diazepam,Allopurinol, Phenobarbital, Naproxen, Propylthiouracil, Chloramphenicol,Trimethoprimsulfamethoxazxole, Amrinone, Disopyramide, Azathioprine,Cimetidine, and Ranitidine.

[0117] Vascular lesions of the liver, including thrombosis of thehepatic veins, occlusion of the hepatic venules or veno occlusivedisease (VOD), and peliosis hepatitis, can be produced by drugs. Inaddition, lesions including sinusoidal dilation, perisinusoidalfibrosis, and hepatoportal selerosis can occur. Midzonal and pericentralsinusoidal dilatation was first reported as a complication of oralcontraceptive therapy. Peliosis hepatitis is a condition consisting oflarge blood-filled cavities that results from leakage of red blood cellsthrough the endothelial barrier, followed by perisinusoidal fibrosis. Ithas been described in patients taking oral contraceptives, anabolicsteroids, azathioprine and danazol. Injury and occlusion of the centralhepatic venules is also known to be related to the ingestion ofpyrrolizidine alkaloids, such as bush teas. The initial lesion iscentral necrosis accompanied by a progressive decrease in venulecaliber. All of these lesions may be only partially reversible when thedrug is stopped and cirrhosis can develop.

[0118] Several types of benign and malignant hepatic neoplasm can resultfrom the administration of hepatotoxic compounds. Adenomas, a lesionrestricted to women in the childbearing years, is related to the use ofcontraceptive steroids and the risk increases with duration of use.Hepatocellular carcinoma may also be seen in patients taking androgenichormones for aplastic anemia or hypopituitarism.

[0119] Hepatotoxic compounds known to cause hepatic liesons include thefollowing: Contraceptive steroids, Pyrriolizidine alkaloids, Urethane,Azathioprine, 6-Mercaptopurine, 6-Thioguanine, Mitomycin, BCNU,Vincristine, Adriamycin, Intravenous Vitamin E, Anabolic-androgenicsteroids, Azathioprine, Medroxyprogesterone acetate, Estrone sulfate,Tamoxifen, inorganic arsenicals, Thorium dioxide, Vitamin A,methotrexate, Methylamphetamine hydrochloride, Vitamin A,Corticosteroids, Thorium dioxide, and Radium therapy.

[0120] Liver damage caused by other factors usually takes similar forms.Liver damage, whether caused by the hepatotoxicity of a compound,radiation therapy, genetic predisposition, mechanical injury or anycombination of such and other factors, can be detected by several means.Biochemical tests have been used clinically for many years as thestandard measure of hepatotoxicity. Most biochemical tests generallyfall into two categories: tests which measure specific liver markers,for example, prothrombin clotting time, and/or hepatic blood flow, ortests which analyze serum markers, for detection of necrosis,cholestasis, progressive fibrogenesis, or hepatoma (Cornelius, C. inHepatotoxicology, Meeks et al. eds., pgs. 181-185 (1991)). Theimportance of such tests lies in their simplicity and the fact that theyare non-invasive. The rationale for the use of serum enzymes inassessing liver damage is that these enzymes, normally contained in theliver cells, gain entry into the general circulation when liver cellsare injured.

[0121] Elevated serum enzyme activity suggests nercrosis and/orcholestasis. Elevated levels of serum bilirubin conjugates suggest intraor extra hepatic cholestasis. However, there are certain limitations forthe use of serum enzyme levels as single means of diagnosing liverinjury. Serum enzyme levels may increase as a result of leakage fromcells with altered permeabilitv due to systemic effects of an agentrather than specific liver injury caused by a chemical.Histopathological examination of the liver is the next logical step inidentifying and quantifying the nature and extent of liver injury.

[0122] The serum enzymes as markers of liver injury can be divided intofour groups based on specificity and sensitivity to liver damage(Kodavanti et al., Supra).

[0123] Group I: these enzymes indicate more selectively hepaticcholestasis when elevated, e.g. alkaline phosphatase (AP),5′-nucleotidase (5′-ND), and a-glutamyl transpeptidase (G-GT) andleucine aminopeptidase (LAP).

[0124] Group II: These enzymes indicate parenchymal injury whenelevated, e.g., aspartate transaminase (AST), alanine transaminase(ALT), fructose-1,6-diphosphate aldolase (ALD), lactate dehydrogenase(LDH), isocitrate dehydrogenase (ICDH), ornithine-carbamoyl-transferase(OCT), and sorbitol dehydrogenase (SDH) arginase and guanase.

[0125] Group III: These enzymes represent injury of other tissue whenelevated e.g., creatine phosphokinase (CPK).

[0126] Group IV: These enzymes are depressed in hepatic injury, e.g.,cholinesterase (ChE).

[0127] Other serum markers include, procollagen type III peptide levels(PIIIP) to assess if hepatic fibrogenesis is active; ammonia bloodlevels in hepatoencephalopathies; ligand in levels in necrosis andhepatoma; hyaluronate levels due to hepatic endothelial cell damage;a-1-fetoprotein (AFP) levels to detect hepatoma; carcinoembryonicantigen (CEA) levels to detect cancer metastasis to the liver;elevations of antibodies against a variety of cellular components, suchas, mitochondrial, and nuclear and specific liver membrane protein; anddetection of proteins, such as, albumin, globin, amino acids,cholestrol, and other lipids. Also, biochemical analysis of a variety ofminerals, metabolites, and enzymes obtained from liver biopsies can beuseful in studying specific biochemical defects in inherited, acquired,and experimentally induced liver disorders.

[0128] Liver function tests can be performed to assess liver injury.Liver function tests include the following:

[0129] Group I assessment of hepatic clearance of organic anions, suchas, bilirubin, indocyanine green (ICG), sulfobromophthalein (BSP) andbile acids;

[0130] Group II assessment of hepatic blood flow by measurements ofgalactose and ICG clearance;

[0131] and Group III assessment of hepatic microsomal function, throughthe use of the aminopyrine breath test and caffeine clearance test. Forexample, serum bilirubin can be measured to confirm the presence andseverity of jaundice and to determine the extent of hyperbilirubinemia,as seen in parenchymal liver disease. Aminotransferase (transaminase)elevations reflect the severity of active hepatocellular damage, whilealkaline phosphatase elevations are found with cholestasis and hepaticinfiltrates (Isselbacher, K. and Podolsky, D. in Hartison's Principlesof Internal Medicine, 12th edition, Wilson et al. eds., 2:1301-1308(1991)). Methods for performing serum enzyme analysis are known in theart and are, for example, described in Kodavanti et al. Supra.

[0132] Because extensive liver injury may lead to decreased blood levelsof albumin, prothrombin, fibrinogen, and other proteins synthesizedexclusively by hepatocytes, these protein levels may be measured asindicators of liver injury. In contrast to measurements of serumenzymes, serum protein levels reflect liver synthetic function ratherthan just cell injury (Podolsky, D. Principles of Internal Medicine,12th edition, Wilson et al. eds., 2: 1308-1311 (1991)).

[0133] In many patients, computed tomography (CT), ultrasound,scintiscans, or liver biopsy may be needed to determine the nature ofthe liver disease (Isselbacher, K, Supra and Friedman, L. and Needleman,L. in Harrison's Principles of Internal Medicine, 12th edition, Wilsonet al. eds., 2: 1303-1307 (1991)).

[0134] The present invention provides methods for enhancing the effectof chemotherapy of cancer in a subject, said methods comprisingadministering to the subject a VEGFR modulating agent in a mannereffective to protect the liver of the subject from damage caused by abepatoxic compound prior to, or simultaneous with, the chemotherapy,thereby increasing the subject's tolerance to the chemotherapy. Thechemotherapeutic agents used during the course of chemotherapy can havecytotoxic effects upon hepatic cells, therefore limiting the dosageand/or duration of the chemotherapeutic agent being administered to thepatient. By exposing the liver to a composition comprising a VEGFRagonist such as VEGF, FIt-sel or KDR-sel, such toxic effects can beprevented or reduced. As such, the dosage of the chemotherapeutic agentscan be increased, thereby enhancing the efficacy of the cancer therapy.

[0135] A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin γ₁ ¹ and calicheamicin θ¹ ₁,see, e.g., Agnew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antiobioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; maytansinoids such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY 117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

[0136] Pharmaceutical Compositions and TherapeuticlprophylacticAdministration

[0137] For in vivo uses according to the methods of the invention, atherapeutic compound of the invention is administered to a subject usingmethods and techniques known in the art and suitable for the particularuse. In a preferred embodiment, the compound is administered in the formof pharmaceutical compositions at a pharmaceutically acceptable dosage.

[0138] In one aspect, the invention contemplates the use of mammaliancell preparations for the administration of a therapeutic protein agent(such as VEGF, Flt-sel or KDR-sel). The mammalian cells used herein havebeen transfected with the heterologous gene encoding the protein, asdescribed in detail above. In a preferred embodiment, the host cellsused for the administration are CHO cells.

[0139] In another aspect of the invention, the therapeutic agent can beentrapped in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, in colloidal drug deliverysystems (e.g., liposomes, microspheres, microemulsions, nanoparticlesand nanocapsules), or in macroemulsions. Such techniques are known inthe art and disclosed in Remington, the Science and Practice ofPharmacy, 20th Edition, Remington, J., ed. (2000).

[0140] In one aspect of the invention, the therapeutic agent can beadministered in vivo in slow-release preparations. Suitable examples ofslow-release preparations include semipermeable matrices of solidhydrophobic polymers containing the multivalent antibody, which matricesare in the form of shaped articles, e.g., films, or microcapsule.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for sborter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0141] The therapeutic composition of the invention can be administeredby any suitable means, including but not limited to, parenteral,subcutaneous, intraperitoneal, intrapulmonary, and intranasaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. Inaddition, the therapeutic composition is suitably administered by pulseinfusion, particularly with declining doses of the antibody. Preferablythe therapeutic composition is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic.

[0142] It is further contemplated that a therapeutic protein agent ofthe invention (such as VEGF, Flt-sel or KDR-sel) can be introduced to asubject by gene therapy. Gene therapy refers to therapy performed by theadministration of a nucleic acid to a subject. For general reviews ofthe methods of gene therapy, see, for example, Goldspiel et al. (1993)Clinical Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95;Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan(1993) Science 260:926-932; Morgan and Anderson (1993) Ann. Rev.Biochem. 62:191-217; and May (1993) TIBTECH 11:155-215. Methods commonlyknown in the art of recombinant DNA technology which can be used aredescribed in Ausubel et al. eds. (1993) Current Protocols in MolecularBiology, John Wiley & Sons, NY; and Kriegler (1990) Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY.

[0143] There are two major approaches to getting the nucleic acid(optionally contained in a vector) into the subject's cells; in vivo andex vivo. For in vivo delivery the nucleic acid is injected directly intothe subject, usually at the site where the protein is required. For exvivo treatment, the subject's cells are removed, the nucleic acid isintroduced into these isolated cells and the modified cells areadministered to the subject either directly or, for example,encapsulated within porous membranes which are implanted into thesubject (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). There are avariety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells ex vivo, or in vivo in the cellsof the intended host. Techniques suitable for the transfer of nucleicacid into mammalian cells ex vivo include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retrovirus.

[0144] The currently preferred in vivo nucleic acid transfer techniquesinclude transfection with viral vectors (such as adenovirus, Herpessimplex I virus, lentivirus, retrovirus, or adeno-associated virus) andlipid-based systems (useful lipids for lipid-mediated transfer of thegene are DOTMA, DOPE and DC-Chol, for example). Examples of using viralvectors in gene therapy can be found in Clowes et al. (1994) J. Clin.Invest. 93:644-651; Kiem et al. (1994) Blood 83:1467-1473; Salmons andGunzberg (1993) Human Gene Therapy 4:129-141; Grossman and Wilson (1993)Curr. Opin. in Genetics and Devel. 3:110-114; Bout et al. (1994) HumanGene Therapy 5:3-10; Rosenfeld et al. (1991) Science 252:431-434;Rosenfeld et al. (1992) Cell 68:143-155; Mastrangeli et al. (1993) J.Clin. Invest. 91:225-234; and Walsh et al. (1993) Proc. Soc. Exp. Biol.Med. 204:289-300.

[0145] In some situations it is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein on the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al. J.Biol. Chem. 262:4429-4432 (1987); and Wagner et al. Proc. Natl. Acad.Sci. USA 87:3410-3414 (1990). For review of the known gene marking andgene therapy protocols see Anderson et al. Science 256:808-813 (1992).

[0146] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of a VEGFRmodulating agent, and a pharmaceutically acceptable carrier. In aspecific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, including but notlimited to peanut oil, soybean oil, mineral oil, sesame oil and thelike. Water is a preferred carrier when the pharmaceutical compositionis administered orally. Saline and aqueous dextrose are preferredcarriers when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions are preferably employed as liquid carriers for injectablesolutions. Suitable pharmaceutical excipients include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions suspensions, emulsions, tablets, pills, capsules,powders, sustained-release formulations and the like. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of the Therapeutic,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0147] In a preferred embodiment, the composition is formulated, inaccordance with routine procedures, as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water-free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile water orsaline for injection can be provided so that the ingredients may bemixed prior to administration.

[0148] The therapeutics of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withfree carboxyl groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., those formed with freeamine groups such as those derived from isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc., and those derived fromsodium, potassium, ammonium, calcium, and ferric hydroxides, etc. Theamount of the Therapeutic of the invention which will be effective inthe treatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. However, suitable dosage ranges for intravenousadministration are generally about 20-500 micrograms of active compoundper kilogram body weight. Suitable dosage ranges for intranasaladministration are generally about 0.01 pg/kg body weight to 1 mg/kgbody weight. Effective doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. Suppositoriesgenerally contain active ingredient in the range of 0.5% to 10% byweight; oral formulations preferably contain 10% to 95% activeingredient.

[0149] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

[0150] The following examples are intended merely to illustrate thepractice of the present invention and are not provided by way oflimitation. The disclosures of all patent and scientific literaturescited herein are expressly incorporated in their entirety by reference.

EXAMPLES Example 1 VEGFR Selective Variants of VEGF

[0151] Generation and characterization of VEGF variants that selectivelybind and activate a specific VEGF receptor (such as KDR or Flt-1) havebeen known in the art and described in, for example, Li et al. (2000) J.Biol. Chem. 275:29823; Gille et al. (2001) J. Biol. Chem. 276:3222-3230;PCT publications WO 00/63380 and 97/08313; and U.S. Pat. No. 6,057,428,the disclosure of which are expressly incorporated herein by reference.

[0152] Specifically, a VEGF variant with high selectivity for the Flt-1receptor was generated by combining four mutations that greatly affectedKDR but not Flt-1 binding. Mutation to alanine of Ile 43, Ile 46, Gln 79or Ile 83 showed that the side chains of these residues are critical fortight binding to KDR but unimportant for Flt-1-binding. Li et al. (2000)supra. A Flt-sel variant was constructed with alanine substitutions atpositions Ile 43, Ile 46, Gln 79 and Ile 83, using site directedmutagenesis methods described by Kunkel et al. (1991) Methods Enzymol.204:125-139. This particular Flt-sel variant can also be represented bythe identifier, I43A/I46A/Q79A/I83A. The corresponding codons for thesefour alanine substitutions at positions 43, 46, 79 and 83 areGCC/GCC/GCG/GCC, respectively.

[0153] Various assays were conducted to examine the properties andbiological activities of the I43A/I46A/Q79A/I83A Flt-sel variant. Li etal. (2000) supra. For example, quantitative binding measurements werecarried out using a soluble radio-immuno receptor-binding assay (RIA).In the assay, native VEGF(8-109) had affinities for KDR and Flt-1 of 0.5nM and 0.4 nM, respectively. Fit-sel was found to have at least 470-foldreduced KDR-binding affinity in this assay. Somewhat surprisingly, sincesmall reductions in Flt-1-binding had been observed from the individualpoint mutants in the ELISA, the FIt-sel variant's affinity for Flt-1 wasessentially identical to that of the native protein.

Example 2 Promotion of Liver Growth

[0154] Methods and Materials

[0155] The full-length cDNA encoding for human VEGF₁₆₅ (Leung et al.(1989) Science 246:1306-9) was inserted into the dicistronicdihydrofolate reductase (DHFR) intron expression vector SV.DI (Lucas etal. (1996) Nucleic Acids Res. 24:1774-9), resulting in the constructSV.DI.VEGF₁₆₅.H8. A control construct, SV.DI.HAg.H8, was similarlyconstructed using cDNA encoding for Hakata antigen (HAg) as insert. HAgis a serum glycoprotein which has been recently shown to be anautoantigen in systemic lupus erythematosus patients. Sugimoto et al.(1998) J. Biol. Chem. 273:20721-7. This protein has structural homologyto the angiopoietin family, although it does not interact with the The-2receptor.

[0156] Using either construct described above, a dicistronic mRNA wastranscribed under the control of the SV40 early gene promoter andencoded both the DHFR gene and the inserted gene of interest, which werethereby expressed in a fixed equimolar ratio. Both open reading frameswere also fused with a DNA sequence encoding for a short carboxylterminal peptide tag consisting of 8 histidine residues allowing easydetection and purification. The linearized expression plasmids,SV.DI.VEGF₁₆₅.H8, and SV.DI.HAg.H8 as well as an empty vector, SV.DI.H8,were electroporated into DHFR—CHO cells. Prior to transfection, thesecells were grown in growth medium consisting of F12/DMEM-based mediumcontaining high concentrations of amino acids and insulin supplementedwith 10% FBS, L-glutamine (2 mM), glycine (10 μg/ml), hypoxanthine (15μg/ml), and thymidine (5 μg/ml)(GHT). Pools of stably transfectedDHFR+CHO cells were derived and maintained by selection in GHT-free richmedium (Lucas et al. (1996) Nucleic Acids Res. 24:1774-9). Expression ofthe VEGF₁₆₅ protein by VEGF₁₆₅ transfected CHO cells (CHO-VEGF₁₆₅) wasconfirmed by immunoblotting and ELISA. A band of ˜23 kDa, whichcorresponds to the expected molecular weight of the VEGF₁₆₅ monomer, wasimmunoprecipitated from conditioned culture media of CHO-VEGF₁₆₅ cells.Such band was not present in culture supernatants of CHO-DHFR controlcells.

[0157] For generation of CHO-HGF cells, the full length cDNA encodingfor HGF was inserted into the pRK5 expression vector and transfectedinto CHO cells, as described previously (Zioncheck et al. (1995) J.Biol. Chem. 270:16871-8). HGF is a key mitogen for hepatocytes and hassequence similarity to plasminogen. Nakamura et al. (1987) FEBS Lett.224:311-6. HGF is secreted as a single-chain 82-84 kDa promitogen whichis endoproteolytically processed into the bioactive HGF heterodimer,consisting of a 69 kDa α-chain and a 32 kDa β-chain. Nakamura et al.(1987) FEBS Lett 224:311-6. A band consistent in molecular weight withthe 69 kDa HGF α-chain was detected in conditioned culture media ofCHO-HGF cells but not in CHO-DHFR controls. Using the control constructSV.DI.HAg.H8, another stably transfected CHO cell pool was alsogenerated, which secretes high amounts of HAg. Under reducingconditions, the HAg monomer was detected in CHO-HAg conditioned culturemedia as a band of ˜32 kDa.

[0158] VEGF-PLGA microspheres were used as a slow release formulation ofVEGF. VEGF-PLGA microspheres were generated by encapsulating VEGF₁₆₅protein into poly(lactic-co-glycolic acid) biodegradable polymer(provided by J. Cleland and A. Daugherty, Genentech). About 10% of thetotal VEGF protein contained in the microspheres was released into thecirculation.

[0159] Juvenile (3-4 week old) or adult athymic beige nude xid mice(Hsd:NIHS-bg-nu-xid, Harlan Sprague Dawley (Indianapolis, Ind.)) wereanesthetized by isofluorane inhalation. Each group consisted of fiveanimals. Suspensions of stably transfected or control CHO cells incomplete culture medium were intramuscularly injected into both legs ofthe animal. A total volume of 100 μl cell suspensions containing about3×10⁶ cells was injected into 3 different sites of the anterior femoralmuscles of each leg. On day 14 after injection, the mice weresacrificed, and serum samples were isolated and further analyzed.

[0160] VEGF-PLGA treated animals received a total of 3 intramuscularinjections of 100 μl of PLGA-VEGF microspheres per leg at days 1, 7, and10. The dose of released VEGF per injection was about 4.5 mg/kg. TheVEGF-PLGA injected animals were sacrificed on day 15. One hour beforesacrifice, all animals were intraperitoneally injected with 100 mg/kgbromodeoxyuridine (BrdU) (Sigma, St. Louis, Mo.). Livers, kidneys,heart, legs, and brains were removed and weighed. The collected tissueswere immersed in formalin for histological evaluation.

[0161] For proliferation studies, animals were injected with BrdU (100mg/kg) 1 hour prior to killing. Tissues were fixed in 10% neutralbuffered formalin for 12 to 16 hours prior to paraffin embedding. BrdUimmunohistochemoistry was performed as previously described. Gerber etal., (1999). A monoclonal rat anti-mouse antibody for F4/80 (Serotec,Raleigh, N.C.) was used at 10 μg/ml (1:1000 dilution) on 5 μm paraffinliver sections. The incubation was at 4° C. overnight. This antibodyrecognizes a 160 kD glycoprotein expressed by murine macrophages. Leenenet al. (1994) J. Immunol. Methods 174:5-19. This antigen is notexpressed by lymphocytes or polymorphonuclear cells. To demonstratespecificity, rat IgG 2B (Pharmingen, San Diego, Calif.) was used as anegative control. Biotinylated rabbit anti-rat IgG was used as asecondary antibody. The antibody was detected using Vectastain Elite ABCreagent (Vector Labs, Burlingame, Calif.) followed by Metal Enhanced DAB(Pierce, Rockford, Ill.). The sections were counterstained with Mayer'shematoxylin.

[0162] Nude mice that have been intramuscularly injected with CHO cellswere anesthetized by intraperitoneally injecting 100 μl Nembutal (AbbottLaboratories, Chicago, Ill.) and perfused with 10 ml of perfusion buffer(NaCl 142 mM, KCl 6.7 mM, HEPES 10 mM). After sacrificing the animals,the livers were removed and minced into fine pieces. Collagenasedigestion of the tissue pieces was carried out at 37° C. for 30 min indigestion buffer (67 mM NaCl, 6.7 mM KCl, HEPES 100 mM, CaCl₂ 5 mM)supplemented with 50 μg/ml Liberase RH (Roche Molecular Biochemicals,Indianapolis, Ind.). Single cell suspensions were obtained after passingthe digested liver suspension through a 70 μm cell strainer (Falcon,Bedford, Mass.). For BrdU staining, the in situ Cell Proliferation Kitwas used according to the manufacturer's recommendations (RocheMolecular Biochemicals, Indianapolis, Ind.). In short, cells werewashed, ethanol-fixed and the DNA denatured by HCl treatment.DNA-incorporated BrdU into was detected by staining with ananti-BrdU-FLUOS antibody (anti-BrdU-F(ab′)₂—FITC conjugate). For flowcytometry, brightly autofluorescent monocytic cells were rejected fromthe analysis by forward/side scatter-gating. 10,000 gated cells wereacquired and analyzed on a Becton Dickinson (San Jose, Calif.)FACSCalilbur flow cytometer.

[0163] For the detection of recombinant human VEGF protein in mouseserum samples, a fluorimetric anti-VEGF enzyme-linked immunosorbantassay (ELISA) was performed as described before. Rodriguez et al. (1998)J. Immunol. Methods 219:45-55. The limit of sensitivity of the assay inthe presence of mouse serum was 200 pg/ml. The assay is specific forhuman VEGF and does not cross-react with murine VEGF.

[0164] For detection of recombinant human HGF, a previously describedELISA was used (Koch et al. (1996) Arthr. Rheum. 39: 1566-75), with somemodifications. In brief, an anti-human HGF monoclonal antibody was usedto coat 96-well microtiter plates. After a 1-hour incubation at roomtemperature, the wells were washed and serial dilutions of serum sampleswere added. rHGF was used as a reference standard. Following a 2-hourincubation and a wash, biotinylated sheep anti-HGF was added andincubated for 1 hour. After washing, horseradish peroxidase conjugatedstreptavidin (Amdex) was added and incubated for 30 minutes. Afterwashing, the substrate solution, tetramethyl benzidine (Sigma), wasadded. Plates were read on a microtiter plate reader (Molecular Devices)at 450 nm with a subtracted blank at 650 nm. A four-parameter curvefitting program was used to generate a standard curve and sampleconcentrations were derived by interpolation in the standard curverange.

[0165] All animals were injected with BrdU (100 mg/kg) 1 hour prior tokilling. Tissues were fixed in 10% neutral buffered formalin for 12 to16 hours prior to paraffin embedding. H&E staining andimmunohistochemisty for Flk-I expression was performed as describedpreviously (Gerber et al., 1999). Briefly, tissue sections werepretreated with Trilogy antigen retrieval solution (Cell Marque, Austin,Tex.) at 99° C. for 1 hr and then incubated with rat anti-mouse Flk-1(mab MALK-1, Genentech) at 3.9 mg/ml. overnight at 4° C.Immunoreactivities were visualized by the avidin-biotin complextechnique using Vectastain Elite ABC kit (Vector Laboratories,Burlingame, Calif.) with diaminobenzidine as chromogen. Hematoxylin wasused as counterstain. BrdU immunohistochemoistry was performed aspreviously described (Gerber et al., 1999).

[0166] Results

[0167] Transplant of CHO-VEGF Cells Results in Liver Growth In Vivo

[0168] To study the effect of sustained levels of VEGF protein in mice,CHO-VEGF cells were injected intramuscularly into both legs of 3-4 week,6-8 week or 12-14 week old beige nude mice. An identical number ofCHO-DHFR cells, CHO-HAg cells, or CHO-HGF cells was injected intocontrol animals. After two weeks, the serum concentrations of human VEGFin the CHO-VEGF animals were 3.3+1.7 ng/ml, (range 0.8-5.4 ng/ml). hVEGFwas undetectable in the sera of CHO-DHFR and CHO-HAg control animals.The HGF levels in the serum of CHO-HGF animals were 1.25±0.87 ng/ml(range 0.50-2.00).

[0169] As shown in FIGS. 1A-1D, substantially increased liver sizes wereobserved in the CHO-VEGF groups. Very similar results were obtained inboth age groups and therefore only data from the 3-4 week old group wereshown. Since the brain weight remained unchanged within treatment groups(FIG. 1D), other organ weights were normalized to the constant brainweight. The liver/brain ratio, i.e. the relative liver mass, of theCHO-VEGF group (4.73±0.39) was highly significantly increased whencompared to the CHO-DHFR (3.18±0.25, p<0.0001) and the CHO-HAg(3.00±0.45, p<0.0001) controls (FIG. 1A, B). This reflects an increaseof relative liver masses of 49 and 59%, respectively. The heart/brainratio was also significantly increased in the CHO-VEGF group(0.376±0.052 compared to 0.304±0.022 and 0.304±0.017 in the CHO-DHFR andthe CHO-HAg groups, respectively; CHO-VEGF versus CHO-DHFR, p<0.05). Aswell, the kidney/brain ratio (0.976±0.071 compared to 0.860±0.070,p<0.05 and 0.822±0.097, p<0.02 in the CHO-DHFR and the CHO-HAg groups)was significantly increased (FIG. 1C). These effects, however, were muchless pronounced than the increases in liver size. The highly significantincreases in the absolute and relative liver mass in the VEGF-treatedanimals were consistently observed in five independent experiments.Among those different experiments, the relative liver weight wasincreased 30-69% in the CHO-VEGF group when compared to the CHO-DHFRgroup. A further increase in liver mass was observed three weeks afterimplantation of CHO-VEGF cells, but this was frequently associated withsigns of distress and mortality. Interestingly, no appreciable effect onliver growth was observed in the CHO-HGF group (FIG. 1B).

[0170] VEGF in Slow Release Preparation also Promotes Liver Growth

[0171] The above described effects were also reproduced by injecting aslow release formulation of highly purified human recombinant VEGF₁₆₅protein. Mice were i.m. injected in the legs with VEGF-PLGA microsphereson days 1, 7, and 10. The dose of released VEGF protein was about 4.5mg/kg/animal. As in the CHO-VEGF treated mice, the liver/brain ratio ofthe VEGF-PLGA injected mice was significantly increased (4.057±0.274,p<0.05, n=3) when compared to control animals (3.396±0.302, n=4) (FIG.1E). Such increase in liver growth is less pronounced than that obtainedwith the CHO-VEGF cell transplant. However, the concentrations of humanVEGF in the serum of VEGF-PLGA treated animals at the time of sacrificewere 200 pg/ml. This finding indicates that purified recombinant VEGF isalso able to promote liver growth.

[0172] Systemic VEGF Results in a High Number of Mitotic Cells in theLiver

[0173] Standard histological analysis of the livers of CHO-VEGF injectedanimals revealed that a large number of hepatic cells displayed mitoticfigures, as indicated by BrdU immunohistochemistry. Mitotic activity wasseen in both parenchymal and non-parenchymal cells of CHO-VEGF treatedlivers. In livers of CHO-DHFR control animals, only 1 mitotic hepatocytewas seen in only 1 out of 5 animals in a total number of 10 high power(40×) fields examined. In contrast, 100% of the livers of the CHO-VEGFgroup showed at least 5 mitotic figures per 10 high power fields (range5-11). Moreover, the proliferating hepatocyte compartment represented6.44±0.96% (CHO-DHFR 1.02±0.74 and CHO-HGF 1.55±1.48%) as quantified byFACS analysis of hepatocytes isolated from BrdU-injected CHO-VEGF mice.

[0174] Since VEGF is known to be able to promote vascular permeability(Dvorak et al. (1995) Am. J. Pathol. 146:1029-39), the possibility exitsthat at least some of the increase in liver size is due to fluidretention, resulting in hepatocyte swelling. However, analysis of thearea density of hepatocytes did not reveal any difference betweenCHO-DHFR and CHO-VEGF groups, indicating that hepatocyte mitogenesisfully accounts for the effect.

[0175] More Complex Branching of Sinusoid Endothelial Cells in the LiverFollowing VEGF Exposure

[0176] Increased hepatocyte mitotic activity, sinusoidal cellhyperplasia and increased extramedullary hematopoietic activity werepresent in all animals injected with CHO-VEGF cells. However, noevidence of angioma or other abnormal vascular proliferation wasdetected in any specimen. The liver of animals injected with CHO-DHFR,CHO-HAg and CHO-HGF were within normal limits. Immunohistochemistry forFlk-1 around a terminal hepatic venule demonstrated a normal pattern ofsinusoidal and non-sinusoidal endothelial staining in the liver of aCHO-DHFR animal. In the CHO-VEGF liver, the sinusoids appeared to have amore complex branching architecture and an apparent increasedendothelial staining.

Example 3 Stimulation of Hepatocyte Mitogenesis Methods

[0177] Hepatocytes were isolated from nude mice following retrogradeliver perfusion, according to a previously described procedure. Harmanet al. (1987) J. Pharmacol. Methods 17:157-63. The inferior vena cavawas cannulated with a 22G Abbocath T catheter (Abbott Lab) and theportal vein was severed. The livers were perfused in situ with a 0.1%collagenase, 2 mM CaCl2 in PBS solution at a flow rate of 3 ml/min for10-15 minutes. Hepatocytes were seeded in 24-well plates at a density of5×10⁴ cells per well in William's E medium (GIBCO BRL) supplemented with10% heatinactivated fetal bovine serum (GIBCO BRL), 1 μg/ml insulin, 10μg/ml transferrin, 1 μg/ml aprotinin (Sigma), 2 mM 1-glutamine, 100 U/mlpenicillin and 100 μg/ml streptomycin (GIBCO BRL) and allowed to adhereovernight. The medium was then carefully removed from each well andmedium containing growth factors (murine EGF, murine HGF, recombinanthuman VEGF, murine PIGF, VEGF-E, KDR^(sel) or Flt^(sel)) was added.After a 24-hour incubation, cells were pulsed with 1 μCi/well methyl-3Hthymidine (47 Ci/mmol, Amersham Pharmacia Biotech), and incubatedovernight. The following day, plates were harvested by rinsing in coldPBS followed by a 15 minute incubation in cold 10% TCA. Wells weresubsequently rinsed with water and 200_l of 0.2 N NaOH was added. Thisvolume was then added to scintillation fluid and analyzed in a BeckmanLiquid Scintillation System.

[0178] For trans-well cultures, 5×10⁴ hepatocytes were placed in theupper chamber on a 0.4 _m pore size polyester membrane (Costar), and1×10⁵ LSEC were seeded in the lower chamber of the 24 well plate thatwas previously coated with a 0.002% solution of fibronectin (F1141,Sigma). Cells were allowed to adhere overnight. Media was removed andgrowth factors were added in CSC media (Cell Systems) supplemented with0.2% FCS and 0.1% BSA. After a 24-hour incubation, cells were pulsedwith 1 μCi/well methyl-3H thymidine and incubated overnight.Incorporated counts were assessed as described above.

[0179] Sinusoidal endothelial cells were isolated from nude or C57B16mice. Following retrograde perfusion as above described, the liver wasremoved, minced and parenchymal cells were depleted following 2low-speed centrifugations. The remaining non-parenchymal cells wereincubated with endothelial cell-specific anti-CD31 antibody conjugatedto biotin (Pharmingen MEC13.3) in PBS, 2 mM EDTA and 0.5% BSA, on icefor 5-10 minutes, washed, and then incubated with 25 μl ofstreptavidin-conjugated magnetic microbeads (Milteny biotech). Thestreptavidin-decorated endothelial cells were then captured onLS+/VS+columns placed in the magnetic field of a Vairo MACS separator(Milteny biotech). Cells were washed and then eluted by removing thecolumn from the magnetic field. The identity and purity of endothelialcells was verified by FACS analysis for CD31, CD34 (Pharmingen) andFlk-1 (Genentech), and uptake of Dil-labeled Ac-LDL (BiomedicalTechnologies Inc., Stoughton, Mass.). The purified endothelial cellswere plated in 6-well or 24-well dishes previously coated with a 0.002%solution of fibronectin in CSC-medium with serum and growth factors(Cell Systems), supplemented with an additional 5 ng/ml recombinanthuman VEGF.

[0180] Primary LSEC passage 1 were plated at a density of 1×10⁶/well in6-well plates. After an overnight incubation cells were starved in CSCmedia containing 0.2% FCS, 0.1% BSA for 12-18 h. Media was changed toCSC media containing 0.1% BSA for 90 minutes and then factors (20 ng/ml)were added for a 5 minute incubation. Samples were quickly rinsed incold PBS and lysed in 0.8 ml RIPA buffer (150 mm NaCl, 1% Nonidet P40,0.5% sodium orthovanadate, 50 mM Tris pH8.0) containing a proteaseinhibitor mixture (Roche MB 1836145) and phosphatase inhibitor cocktail(Sigma). Anti-phospho-ERK antiserum was purchased from Cell SignalingTechnology and pan-ERK antiserum, from Signal Transduction Laboratories.

[0181] Results

[0182] VEGF is Not a Mitogen for Hepatocytes

[0183] VEGF has been characterized as a mitogen with a targetselectivity largely restricted to vascular endothelial cells (Conn etal., 1990; Ferrara and Henzel, 1989; Plouet et al., 1989). However,recent studies have reported mitogenic effects of VEGF also on certainnon-endothelial cell types, including retinal pigment epithelial cells(Guerrin et al., 1995), and Schwann cells (Sondell et al., 1999).Therefore, it was important to test whether VEGF has any directmitogenic effect on hepatocytes. As illustrated in FIG. 2A, in freshlyisolated mouse hepatocytes, VEGF tested over a broad concentration rangefailed to induce any increase in ³H-thymidine incorporation. Likewise,neither the VEGFR-selective VEGF variants Flt^(sel) and KDR^(sel), northe naturally occurring VEGFR-selective agonists PIGF (for VEGFR-1) andVEGF-E (for VEGFR-2) induced hepatocyte proliferation. In contrast, HGFinduced a dose-dependent stimulation, with a maximal increase at ˜50ng/ml. EGF, tested at the concentration of 10 ng/ml, also induced asignificant increase in ³H-thymidine uptake. This is consistent with insitu ligand binding studies showing that VEGF binding sites arelocalized to endothelial cells, but not hepatocytes, in liver sections.Jakeman et al. (1992) J. Clin. Invest. 89:244. Thus, the hepatocytegrowth-promoting effects of VEGF require the action of endothelialcell-derived paracrine mediator(s).

[0184] Co-Culture of Hepatocytes with Sinusoidal Endothelial Cells

[0185] To further probe the molecular mechanism of the hepatocytemitosis, cultures of primary hepatocytes and primary sinusoidalendothelial cells (LSEC) were established either in isolation or in aco-culture system in a trans-well format.

[0186] In both isolated and trans-well co-cultures, VEGF, KDR^(sel) andVEGF-E induced a 2-2.4-fold increase in 3H-thymidine incorporation inprimary LSEC cultures (FIGS. 2B and 2C), and a robust activation ofERK1/2 phosphorylation as assessed with the phospho-specific antibody(FIG. 3, upper panel). In contrast, Flt^(sel) or PIGF wereindistinguishable from negative control in LSEC proliferation (FIGS. 2Band 2C) and ERK1/2 phosphorylation (FIG. 3, upper panel). HGF had littleeffect on LSEC proliferation (FIG. 2B). This is in agreement withprevious studies showing that HGF is a more potent mitogen forhepatocytes compared to nonparenchymal cells such as LSEC (Patijn et al.(1998) Hepatol. 28:707-16), although in other biological contexts HGF ishighly effective endothelial cell mitogen (Rosen and Goldberg (1997)Rosen, E, Goldberg, ID, Eds. Springer Verlag. pp. 193-208).

[0187] However, in the trans-well format, wt-VEGF, Flt^(sel), or PIGF,resulted in hepatocyte proliferation comparable to that induced byrecombinant mHGF (FIG. 2D). Since these molecules are devoid of directmitogenic effects on hepatocytes (FIG. 2A), these findings indicate thatLSEC are stimulated to release paracrine factors in response to theseligands. Neither KDR^(sel) nor VEGF-E resulted in any significanthepatocyte stimulation, indicating that VEGFR-2 activation is lessefficient at triggering such paracrine effects, at least in LSEC (FIG.2D).

[0188] HGF is a key hepatocyte mitogen and in initial experiments,strong upregulation of HGF mRNA expression were seen by in situhybridization within the sinusoidal endothelium of mice implanted withCHO-VEGF relative to controls. Thus, HGF was tested as one of thepotential paracrine mediators of the VEGF effect in LSEC-hepatocyteco-cultures. Addition of a polyclonal antibody raised against human HGF,which was able to achieve a ˜50% neutralization of the activity of 50ng/ml recombinant murine HGF at the concentration of 50 μg/ml,significantly inhibited the increase in ³H-thymidine incorporationinduced by VEGF (30±2%), Flt^(sel) (29±2.4%) or PIGF (30±1.3%) in theco-culture system. This less than complete inhibition likely reflectsnot only the partial HGF-neutralization, but also the presence ofadditional paracrine factors produced by LSEC.

Example 4 Differential Induction of Hepatotrophic Genes

[0189] To further define the factors induced within the endothelium,potentially contributing to the paracrine effects as previouslydescribed, the levels of RNA transcripts for a number of cytokines andreceptors were examined in primary LSEC. Confluent primary endothelialcell cultures were dissociated by exposure to trypsin and seeded in6-well plates at the density of 2×10⁶ cells/well in growth factor-freeCSC medium with 2.5% FBS. After 10-12 hours, media were changed andcells were incubated for 24 hours with recombinant factor, includingrhVEGF, KDR^(sel), Flt^(sel), mPIGF and VEGF-E, all at 10 ng/ml. Cellswere washed twice in ice cold PBS and total RNA was isolated using theRNeasy kit (Qiagen) according to the instructions of the manufacturer.Fifty ng of total RNA per reaction were analyzed using the RT-PCR kitfrom Perkin-Elmer, following the manufacturer's instructions (PE AppliedBiosystems, Foster City, Calif.). Reactions were run in 96-well platesin a Model 7700 Sequence Detector (PE Applied Biosystems), and resultswere analyzed using Sequence Detection Software (PE Applied Biosystems).RT-PCR conditions were 30 min at 48°, 10 min at 95° C., and 40 cycles of30 s at 95° C. and 90 s at 58° C. Data were normalized to GAPDH level,and total liver RNA was used to generate all standard curves. Eachsample was analyzed in duplicate and the experiments were replicatedtwice for the full set of genes, or five times for HGF.

[0190] Primers and probes used were as follows: TABLE 2 Gene ForwardReverse Probe mGAPDH 5′ATG TTC CAG TAT 5′GAA GAC ACC AGT FAM-AAG CCC ATCACC GAC TCC ACT CAC G AGA CTC CAC GAC A ATC TTC CAG GAG CGA GA-TAMARAHGF GGC AAG GTG ACT CAC ATG GTC CTG FAM-TTT CAG CCC CAG TTG AAT GA ATCCAA TC CAC ATA ACT CAG A- TAMARA HB-EGF TGC TGC CGT CGG ACC GGT CAC CAAFAM-TGA AGC TCT TTC TGA TG CGC G TGG CCG CAG TGT TG- BHQ IL-6 TCC TACCCC AAT TGA ATT GGA TGG FAM-AAC AGA TAA GCT TTC CAA TGC TCT TGG TCC GGAGTC ACA GAA GGA GTG GCT A-BHQ CTGF TTG GCC CAG ACC GGC GCT CCA CTC FAM-TGC GAG CCA ACT CAA CTA TG TGT GGT GCC TGG TCC- BHQ TGF ACC CTG GTG GTAGGG GTC TCC CAA FAM-TGT CAG AGC CTC TAC TGA GAC A GGA AAG ACC GCG ACTC - TAMARA aFGF TGA CGA CTT TTC ACA AGG AGG CTA FAM-AGT TTC CAT TCA TGGATG GA CTG AGA AAG G CCA TTA GGA GGG AGT - BHQ bFGF CCT CTC AGA GAC GGAGGT CAA GGC FAM-CGG TCC AGG TCT CTA CGT TCA A CAC AAT TCC ACC AAC TG -TAMARA PlGF GCA GTA GCC CGT CGG TCC AGG TCT FAM-ACA CAC AAC CCA GGA CTTTCC ACC AAC TG GAC TTG TAT CGG TCA - TAMARA Flt-1 GTC AAC GGC TGC CCGAGC GAT TTG FAM-TCT CTC CCG TGC CCT ATG AT CCT AGT TT AAA CTC CCA CTTG - BHQ Flk-1 TCA TTA TCC TCG CCT TCA TTG GCC FAM-TTC TGG CTC CTT TCGGCA CTG CGC TTA A CTT GTC ATT GTC CTA CGG -BHQ c-Met GCC CTT TCC AGA CATCTC ACT GGC FAM-CCT ATG GAC TAC GAC TTG TT CTG TTC TC CAC TGC CTA GGGGA - TAMARA

[0191]FIG. 4 shows differential induction of hepatotrophic genes by VEGFor VEGFR selective agonists. The panel of potential gene targetsanalyzed included HGF, heparin-binding EGF (HB-EGF), IL-6, connectivetissue growth factor (CTGF), TGFα, TGFβ, aFGF, bFGF, PIGF, Flt-1, Flk-1,and c-Met. Most striking was the reproducible and specific 5.5±2.3-foldinduction of HGF in the VEGF and Flt-selective VEGF treated cultures,indicating that HGF is a target gene for Flt-1 mediated signalingevents. IL-6 also appeared to be a selective target of Flt-1 signaling,induced 3.3±0.6-fold above non-treated LSEC cultures. HB-EGF and CTGFwere induced to equivalent levels by VEGF, Flt^(sel) or KDR_(sel), andtherefore may represent overlapping targets of VEGFR-1 and VEGFR-2signals. In two independent experiments, TGFα, PIGF and Flk-1 werehigher in KDR^(sel) treated cultures. These genes may thereforerepresent VEGFR-2 responsive targets. Although the expression levels ofother transcripts including TGFα, acidic and basic FGF were notincreased by any of the treatments, the levels of these transcripts weresubstantial in the cultured LSEC (i.e. Taqman threshold concentrationvalues less than 23).

Example 5 Gene Delivery of VEGFR Agonists

[0192] To confirm that the effects of VEGF upon liver growth were notdependent upon the mode and/or site of production of VEGF, and tofurther explore the mechanism of VEGF activity, adenoviral vectors wereused to introduce wt-VEGF (Av-VEGF) or receptor-selective agonists(Av-KDR^(sel) or Av-Flt^(sel)) (Gille et al., 2001). The liverrepresents the major organ responsible for blood clearance of Adenovirusand is a natural site of infection via the IV route of administration.

[0193] Ad-VEGF, Av-Flt^(sel) and Av-KDR^(sel) and Ad-lacZ were generatedusing the AdEasy adenoviral vector system (Stratagene) essentially asdescribed by the manufacturer. The coding regions were cloned betweenthe NotI and HindIII sites of the pShuttleCMV vector. These vectors,along with the supplied pShuttleCMV-lacZ, were recombined, in BJ5183electrocompetent bacteria (Stratagene), with the AdEasy vectorcontaining the AdS genome deleted for E1 and E3 regions. Primary viralstocks were prepared by transiently transfecting the recombined AdEasyplasmids into host HEK293 cells. Adenovirus stocks were furtheramplified in HEK293 cells and purified using the Virakit Adenopurification kit (Virapur; Carlsbad, Calif.). Adenovirus titers wereobtained by agarose-overlayed plaque assays.

[0194] Adenovirus was directly injected into the tail vein of mice.Virus was stored in Kit Formulation Buffer supplied by Virapur and theappropriate dilutions were made with PBS. The volume of virus and PBSinjected was 100 μl for each animal. Doses of virus administered were asfollows: Av-VEGF 10⁷ Av-LacZ 5×10⁸, Av-Flt^(sel) 5×10⁸ and Av-KDR^(sel)5×10⁸. Serum was collected when experiments were terminated, 7 or 14days with virus alone, and 6 or 10 days for the CCl₄ experimentsdescribed below. One hour prior to sacrifice, all animals wereintraperitoneally injected with 100 mg/kg bromodeoxyuridine (BrdU)(Sigma, St. Louis, Mo.). Livers, kidneys, heart, legs, and brains wereremoved and weighed. The collected tissues were immersed in formalin forhistological evaluation. Statistical analysis was performed by ANOVA.

[0195] Within one week following injection of Av-VEGF, the liver massincreased by an average of 33.5+/−18.1% (from 23-54%). A clearangiogenic response was seen at a dose as low as of 10⁷ pfu. However,even small increments in the dose of Av-VEGF above 10⁷ were notwell-tolerated and were associated with toxicity and in many casesmorbidity by day 4 post-injection.

[0196] In addition to Av-VEGF, Adenovirus encoding KDR^(sel) orFlt^(sel) were also used for animal injections. Delivery of eachadenovirus resulted in comparable levels of recombinant proteins. Sevendays after delivery of 10⁸ pfu, plasma concentrations of KDR^(sel) andFlt^(sel) were respectively 15±8 and 31±18 ng/ml (n=6). The Av-KDR^(sel)virus elicited a 22.3±7.6% (range 15-30%) increase in liver mass withinone week. Although the increase in mass was of lesser magnitude, themorphological changes induced by Av-KDR^(sel) were qualitativelyindistinguishable from those induced by Av-VEGF and were characterizedby hyperplasia of endothelium lining large vessels and sinusoids, withfocal sinusoidal dilatation. In addition, there was reduplication ofhepatocyte plates, an indicator of recent hepatocyte regenerativeactivity, and in the Av-VEGF group, there was some extramedullaryhematopoiesis. Av-Flt^(sel) VEGF resulted in a small but reproducible(average 5%) increase in liver mass. However, there was no evidence ofangiogenesis in these animals as determined by histological evaluation.Therefore, although the Flt^(sel) was able to stimulate hepatocyteproliferation in vitro, without an accompanying increase in the vascularcompartment in vivo, the overall organ growth was substantiallyattenuated or constrained, indicating that stimulation of angiogenesismay be necessary for maximal growth of adult liver. No liver growth wasassociated with tail vein injection of the control virus, Av-LacZ, atany of the doses tested (up to 10⁹ pfu) and the liver histology wasessentially normal.

[0197] To further investigate the underlying mechanism of liver growth,proliferating cells were counted by assessing bromodeoxyuridine (BrdU)immunohistochemistry, in liver sections 10 days after Av-KDR^(sel) orAv-Flt^(sel) delivery. As shown in FIGS. 5A and 5B, Av-Flt^(sel)promoted a significant increase in hepatocyte proliferation comparedwith Av-KDR^(sel) and Av-LacZ (FIG. 5A). Conversely, Av-KDR^(sel)induced the greatest proliferation of sinusoidal cells (FIG. 5B). TheAv-Flt^(sel)-treated livers showed few proliferating sinusoidal cellsand in this respect were almost indistinguishable from the Av-LacZcontrols (FIG. 5B).

Example 6 Protection Against Liver Damage

[0198] In initial liver protection experiments, adult nude mice wereimplanted with CHO-DHFR or CHO-VEGF cells as above described. After tendays, both groups were subdivided into two subgroups (n=7) according tothe materials the animals receive: vehicle (olive oil) or the potenthepatotoxic agent carbon tetrachloride CCl₄. Both vehicle and CCl₄ weregiven at 4 ml/kg by oral gauvage. After 48 hours, animals were killed,blood was collected and tissues were harvested and fixed as abovedescribed. Five μm paraffin sections were obtained from formalin fixedlivers of CC₄-treated CHO-DHFR (n=7) and CHO-VEGF (n=7) animals. Thesections were stained with Gil's Hematoxylin only and coverslippedbefore conducting a blinded analysis. A Hamamatsu Digital Cameraattached to a Nikon Eclipse TE300 microscope captured bright fieldimages and total tissue area and necrotic area for each sample weremeasured using MetaMorph imaging software (Universal Imaging Corp, WestChester, Pa.). Total tissue area was defined as the total area of thesection analyzed minus the area of the vascular lumens and wasapproximately 20 mm² in each sample analyzed. The data is expressed asthe ratio of necrotic area to total tissue area +/−SE.

[0199] The CHO-DHFR animals exhibited features typical of CCl₄ toxicity,with extensive necrosis of hepatocytes around terminal hepatic venules.In CHO-VEGF animals, the extent of necrosis was substantially reduced.Serum levels of alanine aminotransferase (ALT) and aspartateaminotranferase (AST), which are key indicators of the extent of liverdamage, were reduced respectively 85.3% and 66.3% in the CHO-VEGF grouprelative to CHO-DHFR (Table 3). TABLE 3 Protective Effects of VEGF andVEGFR-selective Variants in the CCL₄ Acute Liver Toxicity Model %reduction % reduction Experiment Group in serum ALT^(a) in serum AST^(a)I CHO-VEGF 85.4 66.3 II^(b) Av-VEGF 36.5 37.9 Av-KDR^(sel) 44.5 59.5Av-Flt^(sel) 65.0 78.7 III^(b) Av-VEGF −36.6 −19.3 Av-KDR^(sel) 86.186.5 Av-Flt^(sel) 70.3 68.3

[0200] The CCl₄ experiments were replicated in the Adenovirus-treatedmice in several studies. Adenoviral vector construction and transfectionare described above. The Adenovirus was given 4 or 8 days prior to CCl₄administration. In Av-VEGF treated livers, ALT levels were reduced anaverage 36.5% in one experiment, relative to Av-LacZ. However, it wasdifficult to reproduce such finding and in one experiment ALT levels inthe Av-VEGF group treated with CCl₄ levels were even higher than in theAv-LacZ group. In this case, VEGF delivery was associated with profoundvascular changes, with extensive sinusoidal dilatation and accompaniedby high mortality. These and other related experiments seem to suggestthat although Av-delivered wild-type VEGF is capable of providing aprotective mechanism to hepatocytes, the very narrow dose response,resulting in toxicity or lack of effects with small changes in virustiter, indicates an unattractive, narrow therapeutic window, at leastwhen VEGF is delivered by this modality.

[0201] In light of the differential responses observed in vitro, furtherstudies were conducted to examine the abilities of Av-KDR^(sel) orAv-Flt^(sel) to rescue liver functions in CCl₄ treated mice. WhenAv-KDR^(sel) was administered 4 days prior the CC14, ALT levels werereduced approximately 45% compared to Av-LacZ (Table 3). The protectiveeffect was greater when Av-KDR^(sel) was delivered 8 days before thetoxic injury, approaching 85% reduction in transaminase levels. Animalsin the Av-Flt^(sel) groups also exhibited a marked protection andapproximately 64% reduction in serum ALT levels relative to AvLacZgroups treated with CCl₄. No significant difference was observed whetherAv-Flt^(sel) was administered 4 or 8 days before CC14 (Table 3).

[0202] The morphology study of livers in the group that receivedAdenoviral vectors 8 days before CCl₄ revealed that there was extensiveconfluent perivenular necrosis in Av-LacZ, involving 30-50% of the totalhepatocyte mass. In Av-KDR^(sel) animals, perivenular hepatocytenecrosis was much less severe, ranging from single cell necrosis tolimited areas of confluent necrosis. Peri-portal areas showed changessimilar to those animals that had not received CC14. Av-Flt^(sel)animals had a similar reduction in hepatocyte necrosis and showed amoderate, mixed inflammatory cell infiltrate around terminal hepaticvenules and mild endothelial cell hyperplasia. Endothelial cell changeswere much less striking than in the KDR^(sel) group.

[0203] Thus, although Av-Flt^(sel) did not induce endothelial cellproliferation or a substantial increase in liver mass in adult animals,it was essentially as effective as KDR^(sel) in preserving liverfunction during acute liver damage. Furthermore, Av-Flt^(sel) displayedno apparent toxicities even when the virus was administered at a dose of10⁹ with animals monitored over a 14 day period.

[0204] The liver toxicity studies with CCl₄ confirmed the markeddifferences in the mode of action of KDR^(sel) and Fit^(sel). Althoughdelivery of both molecules resulted in a comparable degree of liversalvage, the lesions appeared strikingly different morphologically,consistent with different protective mechanisms. Interestingly,KDR^(sel) protection showed a time-dependence, with greater protectionwhen the virus was delivered 8 days before the toxic injury. This isconsistent with the hypothesis that the KDR^(sel) protective effectsprimarily depend on endothelial proliferation, which may amplify aparacrine survival-factor cascade. In contrast, Flt^(sel) was equallyeffective at both time points, consistent with a protective mechanismbased on the release of survival/mitogenic factors from nonproliferatingLSECs, with much reduced or absent dependence on angiogenesis and therelease of survival factors from non-proliferating LSEC.

[0205] The Adenovirus studies also suggest that Av-delivered VEGF,although able to induce potently angiogenesis and liver growth, provedto have a very tight dose-response/toxicity such that it resulted ineither a modest salvage of liver function or in frankly detrimentalaffects with only marginal changes in the virus titer. Doses 2-4 foldlower had no effect, whereas, doses 2-4 fold higher, were associatedwith toxicity and morbidity. The systemic toxicity and tight doseresponse of VEGF have been previously noted (Thurston et al. (2000) Nat.Med. 6:460-63; Wong et al. (2001) Proc. Natl. Acad. Sci. USA 98:7481-6).

[0206] Although Av-KDR^(sel) was toxic when given at the highest doses(109), it showed a better safety profile than VEGF, and even plasmaconcentrations significantly higher than those achieved with Av-VEGFwere associated with less toxicity. It is conceivable that the inabilityof KDR^(sel) to induce the full complement of VEGF target genes, whichincludes inflammatory cytokines like IL-6, may account for suchdifference. Conversely, Flt^(sel) did not induce angiogenesis orsignificant growth of the liver, although in the acute liver toxicitymodel, this molecule salvaged hepatocytes and organ function to animpressive extent. Indeed, one of the most striking conclusions of thisstudy is that, following an appropriate signal, quiescent endotheliumcan be instructed to produce factors that can profoundly protect theparenchyma from injury. This is the first evidence that protectiveeffects on parenchymal cells mediated by the endothelium can beuncoupled from stimulation of angiogenesis.

[0207] Given that the known dose-limiting effects of VEGF (e.g.hypotension, edema) (Yang et al., 1998) are associated with VEGFR-2activation (Kliche and Waltenberger, 2001), it is contemplated thatVEGFR-1 agonists such as Flt^(sel) are useful in forming the basis of atherapeutic scheme aimed toward liver protection. The addition of aVEGFR-2 agonist or other angiogenic factor at a lower ratio may resultin a maximal therapeutic benefit, by providing stimulation ofangiogenesis. Alternatively, a VEGF mutant that preferentially activatesVEGFR-1 versus VEGFR-2 might combine optimal characteristics of safetyand efficacy. The potential indications include acute liver damageinduced by various drugs, chemotherapy, or toxins as well as chronicinjury, including cirrhosis.

[0208] Interestingly, although weights of organs such heart and kidneywere also higher in VEGF-expressing animals relative to controls, thiseffect was smaller than in the liver and, importantly, only liver tissuewas shown to exhibit a substantially increased proportion of cells thathad undergone DNA synthesis. It is noteworthy that induction by VEGF orVEGFR-1 agonists of HGF, IL-6 and some other genes identified in thisstudy is not a general response of endothelial cells; HUVEC or murinelung endothelial cells failed to show any induction of such genes.Therefore, such an endothelial dependent paracrine growth promotingmechanism in response to an ubiquitous molecule like VEGF is, at leastin part, restricted to LSEC and may be another facet of the influence ofthe “microenvironment” on organ diversity (Dellian et al. (1996) Am. J.Pathol. 149:59-71). Previous studies have reported the existence of anangiogenic mitogen, with a selectivity for a specific type ofendothelium (LeCouter et al. (2001) Nature 412:877-884). It is temptingto speculate that the vascular endothelium of other organs may betriggered to release tissue-specific growth factors in response to moreselective “keys” than VEGF.

[0209] Finally, recent studies have linked liver organogenesis topotential inductive signals originating in the endothelium, prior toestablishment of blood flow and vascular functions (Matsumoto et al.,2001). It is tempting to speculate that the mechanism described hereinmay, at least in part, provide an explanation for such inductive events.

1 36 1 25 DNA Artificial sequence sequence is synthesized 1 atgttccagtatgactccac tcacg 25 2 25 DNA Artificial sequence sequence is synthesized2 gaagacacca gtagactcca cgaca 25 3 29 DNA Artificial sequence sequenceis synthesized 3 aagcccatca ccatcttcca ggagcgaga 29 4 20 DNA Artificialsequence sequence is synthesized 4 ggcaaggtga ctttgaatga 20 5 20 DNAArtificial sequence sequence is synthesized 5 cacatggtcc tgatccaatc 20 625 DNA Artificial sequence sequence is synthesized 6 tttcagccccagcacataac tcaga 25 7 17 DNA Artificial sequence sequence is synthesized7 tgctgccgtc ggtgatg 17 8 16 DNA Artificial sequence sequence issynthesized 8 accggtcacc aacgcg 16 9 26 DNA Artificial sequence sequenceis synthesized 9 tgaagctctt tctggccgca gtgttg 26 10 21 DNA Artificialsequence sequence is synthesized 10 tcctacccca atttccaatg c 21 11 21 DNAArtificial sequence sequence is synthesized 11 tgaattggat ggtcttggtc c21 12 34 DNA Artificial sequence sequence is synthesized 12 aacagataagctggagtcac agaaggagtg gcta 34 13 20 DNA Artificial sequence sequence issynthesized 13 ttggcccaga cccaactatg 20 14 18 DNA Artificial sequencesequence is synthesized 14 ggcgctccac tctgtggt 18 15 21 DNA Artificialsequence sequence is synthesized 15 tgcgagccaa ctgcctggtc c 21 16 22 DNAArtificial sequence sequence is synthesized 16 accctggtgg tatactgaga ca22 17 18 DNA Artificial sequence sequence is synthesized 17 ggggtctcccaaggaaag 18 18 22 DNA Artificial sequence sequence is synthesized 18tgtcagagcc tcaccgcgac tc 22 19 20 DNA Artificial sequence sequence issynthesized 19 tgacgacttt tctggatgga 20 20 22 DNA Artificial sequencesequence is synthesized 20 acaaggaggc tactgagaaa gg 22 21 27 DNAArtificial sequence sequence is synthesized 21 agtttccatt caccattaggagggagt 27 22 22 DNA Artificial sequence sequence is synthesized 22cctctcagag acctacgttc aa 22 23 18 DNA Artificial sequence sequence issynthesized 23 ggaggtcaag gccacaat 18 24 23 DNA Artificial sequencesequence is synthesized 24 cggtccaggt cttccaccaa ctg 23 25 18 DNAArtificial sequence sequence is synthesized 25 gcagtagccc gtggactt 18 2623 DNA Artificial sequence sequence is synthesized 26 cggtccaggtcttccaccaa ctg 23 27 27 DNA Artificial sequence sequence is synthesized27 acacacaacc cagacttgta tcggtca 27 28 20 DNA Artificial sequencesequence is synthesized 28 gtcaacggct gccctatgat 20 29 20 DNA Artificialsequence sequence is synthesized 29 ccgagcgatt tgcctagttt 20 30 25 DNAArtificial sequence sequence is synthesized 30 tctctcccgt gcaaactcccacttg 25 31 21 DNA Artificial sequence sequence is synthesized 31tcattatcct cgtcggcact g 21 32 19 DNA Artificial sequence sequence issynthesized 32 ccttcattgg cccgcttaa 19 33 30 DNA Artificial sequencesequence is synthesized 33 ttctggctcc ttcttgtcat tgtcctacgg 30 34 20 DNAArtificial sequence sequence is synthesized 34 gccctttcca gagacttgtt 2035 20 DNA Artificial sequence sequence is synthesized 35 catctcactggcctgttctc 20 36 26 DNA Artificial sequence sequence is synthesized 36cctatggact accactgcct agggga 26

What is claimed is:
 1. A method for promoting liver growth in a subject,comprising administering to the subject an effective amount of a VEGFRmodulating agent, whereby the liver mass of the subject is increased. 2.The method of claim 1, wherein the VEGFR modulating agent is a Flt-1agonist.
 3. The method of claim 2, wherein the Flt-1 agonist comprises aFlt-1 selective VEGF variant (Flt-sel) that selectively binds to Flt-1.4. The method of claim 3, wherein the Flt-sel VEGF variant comprises thefollowing amino acid substitutions to a wild type VEGF: I43A, I46A, Q79Aand I83A.
 5. The method of claim 2, wherein the Flt-1 agonist comprisesPIGF or VEGF-B.
 6. The method of claim 2, wherein the Flt-1 agonist is asmall molecule entity that selectively binds to and activates Flt-1. 7.The method of claim 2, further comprising administering an angiogenicagent in the amount effective to promote proliferation of nonparenchymalcells in the liver.
 8. The method of claim 7, wherein the angiogenicagent is a KDR agonist.
 9. The method of claim 8, wherein the KDRagonist is VEGF, a KDR selective VEGF variant (KDR-sel), an agonisticanti-KDR antibody, or a small molecule.
 10. The method of claim 1,wherein the VEGFR modulating agent is administered to the subjectthrough a systemic delivery system.
 11. The method of claim 10, whereinthe systemic delivery system comprises a cell preparation comprisingmammalian cells expressing a recombinant VEGFR modulating agent.
 12. Themethod of claim 11, wherein the mammalian cells are CHO cells.
 13. Themethod of claim 10, wherein the systemic delivery system comprises aslow release preparation comprising purified VEGFR modulating agent anda polymer matrix.
 14. The method of claim 13, wherein the polymer matrixis a microcapsule selected from the group consisting of liposome,microsphere, microemulsion, nanoparticle and nanocapsule.
 15. The methodof claim 1, wherein the VEGFR modulating agent is administered via aliver-targeted gene delivery vector comprising a nucleic acid encodingthe VEGFR modulating agent.
 16. The method of claim 15, wherein theliver-targeted gene delivery vector is a retroviral vector, adenoviralvector, adeno-associated viral vector or lentiviral vector.
 17. Themethod of claim 15, wherein the liver-targeted gene delivery vector is anonviral vector comprising a cationic liposome.
 18. A method of treatinga pathological liver condition in a subject, comprising administering tothe subject a VEGFR modulating agent in a manner effective to alleviatethe pathological liver condition.
 19. The method of claim 18, whereinsaid pathological liver condition is liver failure, hepatitis, livercirrhosis, toxic liver damage, medicamentary liver damage, hepaticencephalopathy, hepatic coma or hepatic necrosis.
 20. The method ofclaim 18, wherein the VEGFR modulating agent is a Flt-1 agonist.
 21. Themethod of claim 20, wherein the Flt-1 agonist comprises a Flt-1selective VEGF variant (Flt-sel) that selectively binds to Flt-1. 22.The method of claim 21 wherein the Flt-sel VEGF variant comprises thefollowing amino acid substitutions to a wild type VEGF: I43A, I46A, Q79Aand I83A.
 23. The method of claim 20, wherein the Flt-1 agonistcomprises PIGF or VEGF-B.
 24. The method of claim 20, wherein the Flt-1agonist is a small molecule entity that selectively binds to andactivates Flt-1.
 25. The method of claim 20, further comprisingadministering an angiogenic factor in the amount effective to promoteproliferation of nonparenchymal cells in the liver.
 26. The method ofclaim 25, wherein the angiogenic factor is a KDR agonist.
 27. The methodof claim 26, wherein the KDR agonist is VEGF, a KDR selective VEGFvariant (KDR-sel), an agonistic anti-KDR antibody, or a small molecule.28. The method of claim 18, wherein the VEGFR modulating agent isadministered to the subject through a systemic delivery system.
 29. Themethod of claim 28, wherein the systemic delivery system comprises acell preparation comprising mammalian cells expressing a recombinantVEGFR modulating agent.
 30. The method of claim 29, wherein themammalian cells are CHO cells.
 31. The method of claim 28, wherein thesystemic delivery system comprises a slow release preparation comprisingpurified VEGFR modulating agent and a polymer matrix.
 32. The method ofclaim 31, wherein the polymer matrix is a microcapsule selected from thegroup consisting of liposome, microsphere, microemulsion, nanoparticleand nanocapsule.
 33. The method of claim 18, wherein the VEGFRmodulating agent is administered via a liver-targeted gene deliveryvector comprising a nucleic acid encoding the VEGFR modulating agent.34. The method of claim 33, wherein the liver-targeted gene deliveryvector is a retroviral vector, adenoviral vector, adeno-associated viralvector or lentiviral vector.
 35. The method of claim 33, wherein theliver-targeted gene delivery vector is a nonviral vector comprising acationic liposome.
 36. A method for promoting hepatocyte proliferationin the liver of a subject, comprising administering to the subject aFlt-1 agonist, in a manner effective to promote hepatocyteproliferation.
 37. The method of claim 36, wherein the Flt-1 agonistcomprises a Flt-1 selective VEGF variant (Flt-sel) that selectivelybinds to Flt-1.
 38. The method of claim 37, wherein the Flt-sel VEGFvariant comprises the following amino acid substitutions to a wild typeVEGF: I43A, I46A, Q79A and I83A.
 39. The method of claim 36, wherein theFlt-1 agonist comprises PIGF or VEGF-B.
 40. The method of claim 36,wherein the Flt-1 agonist is a small molecule entity that selectivelybinds to and activates Flt-1.
 41. The method of claim 36, wherein theFlt-1 agonist is delivered to the nonparenchymal cells of the liver. 42.The method of claim 41, wherein the nonparenchymal cells are sinusoidalendothelial cells.
 43. The method of claim 36, wherein the Flt-1 agonistis administered to the subject through a systemic delivery system. 44.The method of claim 43, wherein the systemic delivery system comprises acell preparation comprising mammalian cells expressing a recombinantFlt-1 agonist.
 45. The method of claim 44, wherein the mammalian cellsare CHO cells.
 46. The method of claim 43, wherein the systemic deliverysystem comprises a slow release preparation comprising purified Flt-1agonist and a polymer matrix.
 47. The method of claim 46, wherein thepolymer matrix is a microcapsule selected from the group consisting ofliposome, microsphere, microemulsion, nanoparticle and nanocapsule. 48.The method of claim 36, wherein the Flt-1 agonist is administered via aliver-targeted gene delivery vector comprising a nucleic acid encodingthe Flt-1 agonist.
 49. The method of claim 48, wherein theliver-targeted gene delivery vector is a retroviral vector, adenoviralvector, adeno-associated viral vector or lentiviral vector.
 50. Themethod of claim 48, wherein the liver-targeted gene delivery vector is anonviral vector comprising a cationic liposome.
 51. A method forprotecting liver from damage in a subject duet to exposure to ahepatotoxic agent, said method comprising administering to the subject aVEGFR modulating agent, wherein said VEGFR modulating agent effectivelyprotects liver from damage.
 52. The method of claim 51, wherein theVEGFR modulating agent is a Flt-1 agonist.
 53. The method of claim 52,wherein the Flt-1 agonist comprises a Flt-1 selective VEGF variant(Flt-sel) that selectively binds to Flt-1.
 54. The method of claim 53,wherein the Flt-sel VEGF variant comprises the following amino acidsubstitutions to a wild type VEGF: I43A, I46A, Q79A and I83A.
 55. Themethod of claim 52, wherein the Flt-1 agonist comprises PIGF or VEGF-B.56. The method of claim 52, wherein the Flt-I agonist is a smallmolecule entity that selectively binds to and activates FIt-1.
 57. Themethod of claim 52, wherein the Flt-1 agonist is administered incombination with an angiogenic factor.
 58. The method of claim 57,wherein the angiogenic factor is a KDR agonist.
 59. The method of claim58, wherein the KDR agonist is VEGF, a KDR selective VEGF variant(KDR-sel), an agonistic anti-KDR antibody, or a small molecule.
 60. Themethod of claim 51, wherein the VEGFR modulating agent is administeredprior to or concurrent with the exposure of said subject to thehepatotoxic agent.
 61. The method of claim 60, wherein the hepatotoxicagent is a therapeutic agent effective for treating a disease.
 62. Themethod of claim 61, wherein the therapeutic agent is a chemotherapeuticagent for treating cancer.
 63. The method of claim 61, wherein thetherapeutic agent is a radiation agent for treating cancer.
 64. Themethod of claim 51, wherein the VEGFR modulating agent is administeredafter the exposure of said subject to said hepatotoxic agent but priorto any detectable liver damage in the subject.
 65. The method of claim64, wherein said subject is accidentally exposed to said hepatotoxicagent.
 66. The method of claim 65, wherein the VEGFR modulating agentthe Flt-1 agonist is administered within about 10 to about 20 hoursafter the accidental exposure.
 67. The method of claim 52, furthercomprising administering an angiogenic agent in the amount effective topromote proliferation of nonparenchymal cells in the liver.
 68. Themethod of claim 67, wherein the angiogenic agent is a KDR agonist. 69.The method of claim 68, wherein the KDR agonist is VEGF, a KDR selectiveVEGF variant (KDR-sel), an agonistic anti-KDR antibody, or a smallmolecule.
 70. The method of claim 51, wherein the VEGFR modulating agentis administered to the subject through a systemic delivery system. 71.The method of claim 70, wherein the systemic delivery system comprises acell preparation comprising mammalian cells expressing a recombinantVEGFR modulating agent.
 72. The method of claim 71, wherein themammalian cells are CHO cells.
 73. The method of claim 70, wherein thesystemic delivery system comprises a slow release preparation comprisingpurified VEGFR modulating agent and a polymer matrix.
 74. The method ofclaim 73, wherein the polymer matrix is a microcapsule selected from thegroup consisting of liposome, microsphere, microemulsion, nanoparticleand nanocapsule.
 75. The method of claim 51, wherein the VEGFRmodulating agent is administered via a liver-targeted gene deliveryvector comprising a nucleic acid encoding the VEGFR modulating agent.76. The method of claim 75, wherein the liver-targeted gene deliveryvector is a retroviral vector, adenoviral vector, adeno-associated viralvector or lentiviral vector.
 77. The method of claim 75, wherein theliver-targeted gene delivery vector is a nonviral vector comprising acationic liposome.
 78. An article of manufacture comprising: a) acontainer; b) a composition contained within said container; and c) alabel on said container instructing uses of said composition forpromoting liver growth; wherein said composition comprises a VEGFRmodulating agent in the amount effective to promote liver growth.
 79. Akit comprising: a) a first container, a label on said first container,and a composition contained within said first container; wherein saidcomposition comprises a VEGFR modulating agent in the amount effectiveto promote liver growth; b) a second container comprising apharmaceutically acceptable buffer; and c) an instruction for using thekit for promoting liver growth.